Mouse spermatogenesis genes related to human male sterility

ABSTRACT

The present invention provides a polynucleotide or a complementary sequence thereof, which polynucleotide is complementary to mRNA transcribed from a human male infertility-associated gene Scot-t, and has one or more mutations selected from the following group: “t” at 870th position is replaced by “g”; and “t” at 1667th position is replaced by “c”, in the DNA sequence of SEQ ID NO:168; various molecular biological materials relating thereto; various molecular biological materials relating to male infertility-associated gene mutations; and various test methods and diagnostic methods using the same.

TECHNICAL FIELD

The invention of this application relates to an assembly (a group ofgenes: MSGs) of genes involved in mouse spermatogenesis (mousespermatogenesis genes: hereinafter sometimes referred to as “MSG”) and adiagnostic system using the MSGs. More specifically, the inventionrelates to respective methods, e.g., toxicity test, mutagenicity test,and genetic diagnosis, which comprise detecting expression modulation,mutation, replacement of amino acid(s), and the like of MSG usingrespective genes and gene materials (purified polynucleotides,polypeptides as gene expression products, antibodies, etc.). The methodsof the invention can be employed as a mutagenicity test and toxicitytest (inclusive of a test relating to influence on reproduction) ofmedicaments and chemicals or as means for detection or environmentalmeasurement of environmental hormones or endocrine disruptors andenvironmental assessment. Furthermore, the genes and genetic materialsof the invention and respective method inventions contribute developmentof medical technologies on diagnosis, therapy and prevention of maleinfertility and development of diagnostic agents, therapeutic medicinesor preventive medicines therefor, or contraceptive medicines.

Moreover, the invention of the application relates to methods fordiagnosing male infertility wherein mutation or expression change ofhuman male infertility-associated genes which are human homologues ofmouse genes contained in the above MSGs; and wherein such a genepolymorphism as test targets. More specifically, the invention relatesto methods for diagnosing male infertility wherein mutantpolynucleotides derived from male infertility-associated genescharacterized by point mutation or gene polymorphism, mutantpolypeptides as the gene products thereof, and methods for diagnosingmale infertility wherein these mutant polynucleotides and mutantpolypeptides are used as targets. Moreover, similarly to the case of theabove mouse MSG, the gene materials based on the information obtainedfrom human homologues contribute to the development of medicaltechnologies on diagnosis, therapy and prevention of male infertilityand development of diagnostic agents, therapeutic medicines, preventivemedicines, or contraceptive medicines there of.

BACKGROUND ART Mouse Spermatogenesis Genes

Since proposal of the concept of endocrine disruptors (hereinaftersometimes referred to as “ED”) (1997), the risk of a low sperm count andimpaired reproductive function to be induced by the ED has widely beenwell known, a sense of fateful crisis of human being has been raised andalso global environmental conservation measures by development of MSDS(material safety data sheet) and PRTR (pollutant release and transferregisters) have been accelerated, so that development of methods fordetecting and measuring ED and confirmation of influence of ED on livingorganisms become urgent problems. Furthermore, the conventional test for“influence on reproduction” with regard to medicaments, chemical agents,chemical products, chemical substances, and the like is a test only forteratogenicity as a measure. However, in order to avoid and sweep awaythe risk of impaired reproductive function to be induced by the ED, itis defective and insufficient to carry out the conventionalteratogenicity test alone; therefore it is necessary to add detection of“mutagenicity for spermatogenesis genes of mammalians or humanhomologous genes thereof” or test for influence on spermatoblasts andprocesses of their differentiation and spermatogenesis (e.g., toxicitytest or assay using expression modulation, mutation, amino acidreplacement, or the like of MSG in a mouse testis or in vitro as ameasure), to the toxicity test including a test for “influence onreproduction”. Thus, it is considered that the detection or test isconsidered to be a compelling problem to be necessarily solved.

However, heretofore, there are not known technologies on the toxicitytest using expression modulation or mutation of spermatogenesis genes asa measure (test for influence on reproduction), the mutagenicity test,ED detection, and the like. The reason is that spermatogenesis genesusable as test targets are not identified.

Moreover, Western developed countries including Japan, it is known thatabout 10% of total married couple have experienced some forms ofinfertility problems and there is a possibility that about a half ofthem is attributed to factors at the male side. Part of the causes ofmale infertility suggested includes endocrine disorders, genetic factorsincluding chromosomal abnormalities, environmental factors, anomaliesincluding enorchima and varicocele (Rubio C, et al. Human Reprod 2001;10: 2084-2092; Lee P A, et al. (2000) J Urol., 164(5), 1697-1701).However, most of the causes of male infertility are insufficientspermatogenesis and aspermatogenesis and the causes of the problems arenot elucidated yet at present (Cram D S, et al. (2001) J Androl 22(5),738-746). Moreover, among the cases of male infertility, there existcases considered to be associated with various inherited factors(Thielemans B F J, et al. Eur. J. Obst Gynec 1998; 81: 217-225), butcausative genes thereof are not always identified. Therefore,establishment of methods for diagnosing, treating, or preventing maleinfertility (e.g., genetic diagnosis and achievement of genetic therapy,drug treatment, and prevention based on a result of such a diagnosis) isa long-awaited problem to be solved, which provides significant goodnews for human being. Furthermore, when it is called to mind that highrates of birth may be accompanied by poverty, starvation andmalnutrition, spread of infectious diseases such as AIDS, and the likein developing countries, development of contraceptives is alsoconsidered to be an important problem.

As mentioned above, in order to achieve effective detection of ED andthe like by toxicity test or mutagenicity test, or to diagnose maleinfertility more reliably, a molecular biological approach targetingspermatogenesis genes is indispensable. However, heretofore, anyspermatogenesis gene (group) usable in such tests has not beenidentified and, as a matter of course, there is no proposal of means forapplying such a gene (group) to various tests.

The invention of this application is accomplished in consideration ofthe above circumstances and an object thereof is to provide mousespermatogenesis genes (MSGs) usable in the toxicity test, themutagenicity test, or genetic diagnosis relating to ED detection and thelike, and purified polynucleotides, polypeptides, antibodies, and thelike as materials for carrying out such tests.

Also, another object of the invention is to provide respective methodsfor toxicity test, mutagenicity test, and genetic diagnosis using MSGs.

Mutation of Human Male Infertility-Associated Genes

As a result of extensive investigations of the relation between humanhomologues of mouse genes belonging to MSGs and male infertility, theinventors of this application have found that specific mutation in humanScot-t gene and protamine genes is involved in male infertility. In thisregard, with regard to the relation between Scot-t gene and protaminegenes and male infertility, the following have been known.

Succinyl CoA:3-oxo acid CoA transferase (OXCT/SCOT) is one of importantenzymes in the energy metabolism of a ketone body and the ketone body isproduced in the lever and transported to peripheral tissues to be usedas an energy source (Mitchell G A, et al. Clin. Invest. Med. 1995; 18:193-216). Succinyl CoA transferase (SCOT) is localized in a mitochondriaof some tissues and catalyzes formation of acetoacetyl CoA bytransferring the CoA moiety from succinyl CoA to acetoacetic acid. Theacetoacetyl CoA is further decomposed into two acetyl CoA moleculescapable of entering tricarboxylic acid cycle (Williamson, D. H. et al.(1971) Biochem. J., 121, 41-47; Tildon J T et al. J Clinic Invest 1972;51: 493-498). Although cDNA of Scot has been cloned from swine and humanheart (Lin, T. W. and Bridger W. A. (1992) J. Biol. Chem., 267, 975-978;Kassovska-Bratinova S, et al. Am J Hum Genet 1996; 59: 519-528), theinventors of this application has hitherto been cloned a novel genenamed scot-t encoding haploid germ cell-specific SCOT from adifferential cDNA library of mouse testis (Koga M, et al. Biol. Reprod2000; 63: 1601-1609). The SCOT-t is an iso-form of SCOT specific to germcells, is present in haploid sperm and sperm mitochondria, and isconsidered to play a specific role also in spermatogenesis and energymetabolism of sperm. According to Northern blotting, Western blotting,and immunohistochemical analysis, expression of mouse scot-t is detectedin testis, especially in late spermatid, but is not detected in theother somatic cells. The nucleotide sequence of mouse scot-t hashomology of 63.4% and 62.7% to Scot of swine and human hearts,respectively and presumed amino acid sequence has homology of 68.0% and67.4%, respectively. One to 39 residues at NH₂ terminal of SCOT-t form asignal sequence targeting mitochondria. Actually in immunofluorescentstain, localization of SCOT-t protein in mitochondria in immobilizedsperm obtained from tail of epididymis is shown (Koga M, et al. BiolReprod 2000; 63: 1601-1609). The amino acid sequence of SCOT-t containsone glutamic acid residue (the amino acid residue in position of 341th),which corresponds to glutamic acid 344 which is known to be conserved inall CoA transferases including SCOT (Rochet J C, Bridger W A, (1994)Protein Sci., 3, 975-81). The inventors of this application have alsocloned and characterized human orthologue of mouse scot-t. Whole codingregion of mRNA of human Scot-t and deduced amino acid sequence showhomology of 75.4% and 75.8% relative to those of mouse scot-t,respectively. Similarly, the inventors have indicated that h-Scot-t is asingle gene having no introns and is expressed specifically in testis(Tanaka H, et al. Mol Human Reprod 2001; 8: 16-23).

There are some reports on importance of mitochondrial enzymes in energymetabolism in motility and functions of sperm (Pascual, M. I., et al.(1996) Biosci. Rep., 16, 35-40; Yeung, C. H., et al. (1996) Mol. Hum.Reprod., 2, 591-596; Ruiz-Pesini, E. et al. (1998) Clin. Chem., 44,1616-1620). The fact that Scot-t is specifically present is consideredto show the presence of a novel metabolic system wherein a ketone bodyis utilized as an energy source for sperm motion. Furthermore, sinceScot-t is specifically expressed in haploid spermatids, it is suggestedthat Scot-t may play some specific role in spermatogenesis.

Moreover, a remarkable reorganization in sperm nuclei occurs at aspermatogenesis stage. In this process, histone is removed and thenuclei undergo replacement by a specific nuclear protein and finallyreplaced by protamine having a high positive charge, whereby it ishighly compressed (Wounters-Tyrou, D. et al. (1998) Biochimie, 80,117-128; Sassone-Corsi P. (2002) Science, 296, 2176-2178). DNAs of humansperm are assembled in head of sperm in a highly condensed state by twokinds of protamines, i.e., protamine-1 and protamine-2. Protamine-1 is asingle polypeptide molecule having 50 amino acids, on the other hand,protamine-2 is a complex of at least two different forms having 57 and54 amino acids (Mckay, D. J. et al. (1986) Eur. J. Biochem., 158,361-366). Protamine-2 family proteins are synthesized as precursorshaving 66 to 101 residues based on a single copy gene present in the16th chromosome (Krawetz, S. A. et al. (1989) Genomics. 5, 639-645;Reeves, R. H. et al. (1989) J. Hered, 80, 442-446).

It is suggested that male infertility occurs as a result of the nuclearconcentration disorder. In mouse, as a result of early translation ofmRNA of protamine-1, immature nuclear concentration occurs to terminatedifferentiation of sperm (Lee, K. et al. (1995) Proc. Nat.l. Acad. Sci.USA, 92, 12451-12455). In various studies targeting infertile patients,decrease of content of protamine-2 has been reported (Balhorn, R. et al.(1988) Experientia., 44, 52-55; Belokopytova, J. A. et al. (1993) Mol.Reprod. Dev., 34, 53-57), and there is a report that completelyselective protamine-2 deficit is observed in sperm nuclei of part ofmale infertile patients (de Yaba, L. et al. (1993) J. Biol. Chem., 268:10553-10557). However, in the results of sequence analysis ofprotamine-2 gene obtained from these patients, the presence of mutationcausing the detected decrease of protamine-2 is not observed (de Yaba,L. et al. (1993) J. Biol. Chem., 268: 10553-10557; Schlicker, M. et al.(1994) Hum Reprod., 9, 2313-2317). Moreover, it is suggested thatdecrease of protamine-2 occurs owing to incomplete processing ofprotamine-2 precursor molecules in part of infertile patients (d Yebra,L. et al. (1998) Fertil Steril., 69, 755-759).

As mentioned above, it is pointed out that some mutation of Scot-t geneor protamine gene may be associated with male infertility but thereality of the situation is not at all elucidated. An object of theinvention of this application is to provide Scot-t gene mutation andprotamine-2 gene mutation as causal genes of human male infertility.

Also, another object of the invention is to provide a mutant polypeptideexpressed with the above gene mutation, an antibody against the mutantpolypeptide, and a method for diagnosing male infertility using thesemutant gene and mutant polypeptide as test targets.

DISCLOSURE OF INVENTION

This application provides the following mouse spermatogenesis gene groupand inventions utilizing the same as inventions for achieving the aboveobjects.

Namely, the invention provides a group of mouse spermatogenesis genes,which is an assembly of 89 genes in total, wherein the respective genestranscribe mRNAs from which cDNAs having the respective base sequencesof SEQ ID NOs:1 to 89 are synthesized.

The invention provides a cDNA library, which consists of cDNAs derivedfrom the respective genes belonging to said group of mousespermatogenesis genes.

The invention provides a group of DNA fragments each consisting of thebase sequence of continuous 10 to 99 bases of the respective cDNAsbelonging to said group of cDNA library.

The invention provides a group of primer sets for PCR amplification ofDNAs of the respective genes belonging to said group of mousespermatogenesis genes or the respective cDNAs belonging to said group ofcDNAs.

The invention provides a microarray comprising one or more cDNAsbelonging to said group of cDNAs or one or more DNA fragments belongingto said group of DNA fragments.

The invention provides a group of mouse spermatogenesis polypeptides,which is an assembly of 78 polypeptides in total, wherein the respectivepeptides have the respective amino acid sequences of SEQ ID NOs:90 to167.

The invention provides a group of antibodies against the respectivepolypeptides belonging to said group of mouse spermatogenesispolypeptides.

The invention further provides a method for assaying toxicity ormutagenicity of a subject substance, which comprising detectingexpression modulation or mutation of one or more genes belonging to saidgroup of mouse spermatogenesis genes.

Furthermore, the invention provides a method for diagnosing reproductiveability of a subject individual, which comprises detecting expressionmodulation or mutation of one or more genes belonging to said group ofmouse spermatogenesis genes.

Namely, the MSGs of the above invention is cloned by the following threekinds of methods based on the concept that “detection of expressionmodulation and mutation of spermatogenesis genes can be utilized to atoxicity test, especially a reproductive toxicity test and amutagenicity test”.

(a) Cloning Using Monoclonal Antibody

A monoclonal antibody specifically recognizing each processes ofspermatogenesis was prepared and an objective gene (group) wasidentified. Namely, a cell extraction fraction was obtained from mousetestis and rats were immunized therewith. After thorough immunization,the obtained spleen cells were subjected to cell fusion with myelomacells to prepare hybridomas. In order to search for a hybridoma whichprepares an antibody specifically recognizing spermatoblast from theobtained respective hybridomas, the hybridomas were screened by reactingthem with slices of the testis and investigating what kinds of antigenswere recognized by antibodies produced in culture supernatants ofrespective hybridomas, whereby hybridomas producingspermatoblast-specific antibodies were obtained. Using the obtainedmonoclonal antibody, genes encoding antigen proteins recognized by theantibodies were cloned from a mouse testis library expressed inEscherichia coli.

(b) Cloning Using Polyclonal Antibody

A polyclonal antibody specifically recognizing spermatoblasts (inclusiveof cells at all differentiation stage) was prepared and an objectivegene (group) was identified. Namely, a spermatoblast extract fractionwas obtained from mouse testis and rabbits were thoroughly immunizedtherewith. The obtained serum was injected into abdominal cavity of acastrated male mouse and antibodies recognizing cells other thanspermatoblast were absorbed. A serum content of the mouse was collectedand further reacted with a liver extraction fraction. Antigen genesrecognized by the rabbit antibodies contained in the obtained serum werecloned from a mouse testis library expressed in Escherichia coli.

(c) Cloning Using Subtracted Library

A subtracted library containing a concentrated gene group specific tospermatoblasts was prepared and a gene group specifically expressed ateach differentiation stage was cloned, followed by analysis of functionsof each resulting clone. In particular, spermatid is a sole haploid cellwhich is present in an animal individual in a large number for a longperiod of time. Since the genes of the group specifically expressed insuch spermatid each exhibits a specific function for spermatogenesis,these genes of the group were comprehensively cloned and phenomenaspecific to spermatogenesis were analyzed. Specifically, a subtractedlibrary was obtained wherein mRNAs expressed in testis of a 17 day-oldmouse having no spermatid were subtracted from a cDNA library of testisof 35 day-old mouse (C57BL/6) having spermatoblasts at alldifferentiation stages was prepared. From the subtracted library, a genegroup specifically expressed at a morphogenesis stage of spermatid.

By the above methods (a) to (c), 89 clones in total ofspermatoblast-specific gene cDNAs (MSGs cDNAs) were obtained. Then,basic sequences of respective cDNA clones were determined according to aknown method, and it was confirmed that these cDNAs were composed ofbasic sequences shown in odd number sequences of SEQ ID NOS:1 to 89.Moreover, homology search thereof was carried out among various basesequences already reported and it was found that, in 89 of the MSGclone, known genes were about 26%, homologous genes were 32%, andespecially, unknown gene were up to 42%.

Table 1 shows, from left to right, “Sequence Number”, “Name of Gene”(known one), “Database (GenBank) Registry Number”, “Sequence Number ofPolypeptide” encoded by the gene, and “Coding Region” thereof, of 89genes in total shown in SEQ ID NOS: 1 to 89.

TABLE 1 1 AKAP110 AF093406 90  292-2884 2 unidentified 3 Rbcc728(human), Trim36 (human) 91  45-2202 4 Nopp140 (rat) 92 401- 5 93  1-3026 94  12-573 7 95 109-889 8 96 543-861 9 unidentified 10 97  1-372 11 98 1-319 12 ATR AF236887.1 99 168-606 13 100 396-546 14 HSpb (mouse)unidentified 15 unidentified 16 Spergen-1 (rat) AB047508 101  66-513 17102  140-1445 18 103 1046-1994 19 104  362-1127 20 arylsulfatase AX73230 105  642-2160 21 106 111- 22 107  1-202 23 108  47-550 24Drctnnbla 109 228-768 25 110  1-420 26 unidentified 27 111  1-278 28 112 334-2719 29 unidentified 30 113 165-462 31 114 242-695 32 unidentified33 unidentified 34 CDC14B (human) 115  1-534 35 unidentified 36cystatin-related epididymal spermatogenic AF090691 116 180-606 protein37 unidentified 38 117 511-868 39 118  1-619 40 pregnancy-induced growthinhibitor AY037158.1 119 287-698 (human) 41 unidentified 42 120,160-550, 121 618-957 43 fatty acid coenzyme A ligase, long chain 2NM_007981 122   1-2098 44 Fem NM_010193 123  75-1956 45 major 80,000 Mrfibrous sheath component U10341 124  121-2668 46 125  14-566 47 126 46-655 48 Glycerol phosphate dehydrogenase 1, NM_010274 127  131-2312mitochondrial 49 Lim domains containing 1 NM_013860 128  527-2531 50oaz-t AB016275 129 193-788 51 pctp-1 AB031550 130  325-1198 52testis-specific phosphoglycerate kinase M18654 131  21-1272 53phospholipase C delta 4 AF125974 132 26- 54 protamine 1 X07625 133 1-145 55 protamine 2 NM_008933 134  82-388 56 scot-t1 AB022180 135 32-1592 57 scot-t2 AB049996 136  32-1592 58 mitochondrial capsuleselenoprotein NM_008574 137 190-819 59 SP-56 U17108 138  80-181 60Sperizin AB016984 139  113-1093 61 oppo 1 AB074438 140  83-917 62 Galbeta-1, 3-GalNAc-specific GalNAc X93999 141  67-1186 alpha-2,6-sialyltransferase 63 suppressor of fused homolog (Drosophila)NM_015752 142  148-1603 64 t-actin 1 AB023062 143  28-1282 65 t-actin 2AB023063 144  64-1384 66 t-complex Tcp-10a X58170 145  897-2211 67tektin-t AB027138 146  117-1407 68 teek 1 NM_009355 147  28-1129 69 TP-2M60254 148  61-412 70 tsec-1 AB000619 149  211-1252 71 tssk 1.2substrate AF025310 150  25-1603 72 serine/threonine kinase 22B(spermiogenesis NM_009436 151  28-1099 associated) 73 SCP1 D88539 152unidentified 74 tsga2 NM_025290 153  90-942 75 Gapd-S NM_008085 154 1-131 76 meichroacidin D88453 155  90-941 77 halap-X AB032764 156 1-805 78 157  158-1082 79 Ssecks AF326230 158  457-5194 80 gsg1NM_010352 159  144-1056 81 haspin NM_010353 160  34-2296 82 gsg3NM_007605 161  111-1008 83 hils1 NM_018792 162 241-649 84 163  14-120885 164 292-973 86 shippo1 AB067773 165 121-883 87 putativelysophosphatidic acid acyltransferase NM_018743 166  78-534 88 167 1-292 89 unidentified

Furthermore, the following findings (1) to (4) were obtained in theabove MSG clones group.

(1) With regard to the gene structure, most of 89 clones of MSG have nointron and, even if intron is present, most of them have very littleone.

(2) Most of them have no known transcription-related factor-bindingsequence such as TATA, CAAT, GC motif, and the like.

(3) Expression timing is specific to the spermatogenesis stage. Forexample, in the process of primordial germcell->spermatogonium->spermatocyte->spermatid(haploid)->sperm->acquisition of reproductive ability in female genitaltract, there are a number of genes specifically expressing only at thestage of spermatid->sperm.(4) With regard to actions and functions of expressed products, thereare observed those wherein an iso-form characteristic to germ cellagainst somatic cells is present and which exhibits considerablyspecific activity at respective stages of spermatogenesis. For example,those exhibiting the action only in the fertilization process arepresent.

Of these findings, the above (1) and (3) relating to intron areparticularly important. Namely, the reasons are as follows: it issuggested that “the structures and transcription of the genes involvedin spermatogenesis are (1) relatively simple and detection of mutationis not difficult and even when mutation occurs in the gene, the (3)expression occurs only in germ cells and the mutation character does notappear in somatic cells but appears only in germ cells to result ininfertility” and it is judged that detection of the expressionmodulation and mutation is utilizable as a reproduction toxicity testand a mutagenicity test.

In this regard, part of the findings regarding Table 1 and the aboveMSGs are described in detail in the following literatures reported bythe inventors of this application: Int. J. Androl. 20: 361-366, 1997;Gene 204: 159-163, 1997; Genomics 46: 138-142, 1997; Mammal. Genome 8:873-874, 1997; Cytogenet. Cell Gent. 78: 103-104, 1997; Nature 387:607-611, 1997; Dev. Biol. 197: 67-76, 1998; Biol. Reprod 58: 261-265,1998; Gene 237: 193-199, 1999; J. Biol. Chem. 274: 17049-17057, 1999;FEBS Lett. 456: 315-321, 1999; Genomics 57: 94-101, 1999; Biol. Reprod.62: 1694-1701, 2000; Biol. Reprod. 63: 993-999, 2000; Genes Cells 5:265-276, 2000; Biol. Reprod. 63: 1601-1609, 2000; Gene 267: 49-54, 2001;Mol. Human Reprod. 7: 211-218, 2001.

Furthermore, this application provides an invention of mutation of amale infertility-associated genes among the genes contained in the abovegroup of mouse spermatogenesis genes and inventions utilizing the genes'mutation.

Namely, the invention provides a polynucleotide (Scot-t mutantpolynucleotide) or a complementary sequence thereof, whichpolynucleotide is complementary to mRNA transcribed from a human maleinfertility-associated gene Scot-t, and has one or more mutationsselected from the following group:

-   -   “t” at 129th position is replaced by “c”;    -   “t” at 870th position is replaced by “g”;    -   “c” at 1071st position is replaced by “at”; and    -   “t” at 1667th position is replaced by “c”,        in the DNA sequence of SEQ ID NO:168.

The invention provides a Scot-t mutant oligonucleotide or acomplementary sequence thereof, which is part of the above Scot-t mutantpolynucleotide and is a DNA sequence consisting of continuous 10 to 99bases containing the said mutation sites.

The invention provides a polynucleotide (Scot-t mutant genomicpolynucleotide) derived from a human chromosomal DNA, which hybridizesthe above Scot-t mutant polynucleotide or the above Scot-t mutantoligonucleotide or the complementary sequences thereof under a stringentcondition.

The invention provides a primer set for PCR amplification of the aboveScot-t mutant polynucleotide, the above Scot-t mutant genomicpolynucleotide, or the mRNA transcribed from the Scot-t mutant genomicpolynucleotide, wherein one of the primers is an oligonucleotide or acomplementary sequence thereof which is a DNA sequence consisting ofcontinuous 15 to 45 bases containing the mutation site.

The invention provides a polynucleotide (protamine-2 mutantpolynucleotide) or a complementary sequence thereof, whichpolunucleotide is complementary to mRNA transcribed from a human maleinfertility-associated gene protamine-2, and “c” at 248th position isreplaced by “t” in the DNA sequence of SEQ ID NO:173.

The invention provides an oligonucleotide (protamine-2 mutantoligonucleotide) a complementary sequence thereof, which is part of theprotamine-2 mutant polynucleotide and is a DNA sequence consisting ofcontinuous 10 to 99 bases containing the mutation site.

The invention provides a polynucleotide (protamine-2 mutant genomicpolynucleotide) derived from a human chromosomal DNA, which hybridizesthe protamine-2 mutant polynucleotide or the protamine-2 mutantoligonucleotide or the complementary sequences thereof under a stringentcondition.

The invention provides a primer set for PCR amplification of the aboveprotamine-2 mutant polynucleotide, the protamine-2 mutant genomicpolynucleotide, or the mRNA transcribed from the protamine-2 mutantgenomic polynucleotide, wherein one of the primers is an oligonucleotideor a complementary sequence thereof which is a DNA sequence consistingof continuous 15 to 45 bases containing the mutation site.

The invention provides a polypeptide (Scot-t mutant polypeptide), anexpression product of the above Scot-t mutant polynucleotide or Scot-tmutant genomic polynucleotide, which has one or more mutations selectedfrom the following group:

-   -   Leu at 38th position is replaced by Pro;    -   Leu at 285th position is replaced by Arg; and    -   Thr at 352nd position is replaced by Met,        in the amino acid sequence of SEQ ID NO:169.

The invention provides a polypeptide (protamine-2 mutant polypeptide),an expression product of the above protamine-2 mutant polynucleotide orprotamine-2 mutant genomic polynucleotide, which consists of the aminoacid sequence of 1st to 49th positions of the amino acid sequence of SEQID NO:174.

The invention provides an oligopeptide (Scot-t mutant oligopeptide),which is a part of the above Scot-t mutant polypeptide and is an aminoacid sequence consisting of continuous 5 to 30 amino acids containingthe mutation site.

The invention provides an oligopeptide (protamine-2 mutant polypeptide),which is a part of the protamine-2 mutant polypeptide and is an aminoacid sequence consisting of continuous 5 to 30 amino acids.

The invention provides an antibody (anti-mutant Scot-t antibody)prepared using the above Scot-t mutant oligopeptide as an antigen, andan antibody (anti-mutant protamine-2 antibody) prepared using the aboveprotamine-2 mutant oligopeptide as an antigen, respectively.

The invention provides an antibody (anti-protamine-2 antibody) preparedusing the oligopeptide consisting of the amino acid sequence of 50th to91st positions of SEQ ID NO:174 or an amino acid sequence of 1st to 11thpositions of SEQ ID NO:175 as an antigen.

The invention also provides a method for diagnosing male infertility,which comprises detecting presence of the Scot-t mutant genomicpolynucleotide or protamine-2 mutant genomic polynucleotide in achromosomal DNA isolated from a subject person.

In the above diagnostic method, a preferred embodiment comprisesdetecting whether a chromosomal DNA or an mRNA thereof isolated from asubject person hybridizes the Scot-t mutant polypeptide or protamine-2mutant polynucleotide or the Scot-t mutant oligonucleotide orprotamine-2 mutant oligonucleotide, or a complimentary sequence thereofunder a stringent condition or not. Moreover, in the diagnostic method,another preferred embodiment comprises detecting presence of a PCRproduct when PCR is carried out using a chromosomal DNA or mRNA isolatedfrom a subject person as a template with the respective primer sets.

The invention provides a method for diagnosing male infertility, whichcomprises detecting presence of the Scot-t mutant polypeptide orprotamine-2 mutant polypeptide in a biological sample isolated from asubject person.

In the above diagnostic method, a preferred embodiment comprisesdetecting presence of a polypeptide reactive to the anti-mutant Scot-tantibody in a biological sample isolated from a subject person.Moreover, in the diagnostic method, another preferred embodimentcomprises detecting presence of a polypeptide reactive to theanti-mutant protamine-2 antibody but not reactive to theanti-protamine-2 antibody in a biological sample isolated from a subjectperson.

The invention provides a DNA probe, which is labeled Scot-t mutantoligonucleotide or labeled protamine-2 mutant oligonucleotide.

The invention provides a DNA chip comprising the above Scot-t mutantoligonucleotide and/or protamine-2 mutant oligonucleotide.

The invention provides a labeled antibody, wherein the above anti-mutantScot-t antibody, anti-mutant protamine-2 antibody, or anti-protamine-2antibody is labeled.

Namely, as a result of analysis on DNA samples of 516 male persons(infertility: 255 cases, healthy persons: 261 cases) for Scot-t gene and496 male persons (infertility: 226 cases, healthy persons: 270 cases)for protamine genes, the inventors of this application have found thatnucleotide mutations and resulting amino acid mutations as shown inTable 2 are present in each cDNA (Scot-t: SEQ ID NO:168, protamine-1:SEQ ID NO:170, protamine-2: SEQ ID NO:173). They have found that onebase replacement and amino acid mutation in Scot-t and a shortenedprotein caused by one base replacement in protamine-2 are particularlyimportant as a cause of male infertility. In this regard, SEQ ID NO:168corresponds to the base sequence of 4th to 1760th in known human Scot-tcDNA (GenBank/AB050193). SEQ ID NO:170 corresponds to the base sequenceof 532-1089 in known human protamine-1 cDNA (GenBank/M60331). Moreover,SEQ ID NO:173 corresponds to a base sequence of 804-1629 in known humanprotamine-2 cDNA (GenBank/M60332). The positions of nucleotide mutationsand amino acid mutations in Table 2 correspond to the base positions andamino acid positions in the sequence tables. Moreover, the sign of “-”means a non-coding region, “silent” means that the amino acid does notmutate with the nucleotide mutation, and the mark of “***” meansmutation into a stop codon. In addition, “deletion” means deletion of anucleotide and “addition” means addition of a nucleotide.

TABLE 2 Nucleotide Mutation Amino Acid Mutation Scot-t t129c Leu38Prot870g Leu285Arg c1071t Thr352Met t1667c — Protamine-1 c44: eletion —g73: addition — a133g (silent) c160a (silent) g363a (silent) c364a(silent) a431g — Protamine-2 c248t Glu50*** g398c or a — a473c — t493:deletion —

In each invention of this application, a “gene” is present in a genomicDNA and is a double-stranded DNA encoding a specific polypeptide(protein), and it contains a coding region (open reading flame: ORF) andexpression-regulating region(s) (promoter/enhancer sequence, repressorsequence) according to the ORF.

In the invention of this application, the “polynucleotide” and“oligonucleotide” mean long-chain or single-chain nucleotide chains,respectively. A tentative criteria are that the polynucleotide contains100 bp or more and oligonucleotide contains less than 100 bp, but thereexist some exceptions.

In the invention, the “polypeptide” and “oligopeptide” mean long-chainor single-chain peptide chains, respectively. A tentative criteria arethat the polypeptide contains 30 amino acids or more and oligopeptidecontains less than 30 amino acids, but there exist some exceptions.

Moreover, in the following explanation, “influence on reproduction”means not teratogenicity but influence on spermatogenesis and fertility.

Furthermore, the other terms and concepts used in the invention may beexplained in the description of Mode for Carrying Out the Invention andExamples. In this regard, various gene manipulating technologies andmolecular biological technologies used for carrying out the inventionare easily and surely practicable for those skilled in the art based onknown literatures (e.g., Sambrook and Maniatis, in Molecular Cloning—ALaboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989;Ausubel, F. M. et al., Current Protocols in Molecular Biology, JohnWiley & Sons, New York, N.Y., 1995, etc.) except for the technologiesfor which their sources are particularly indicated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration demonstrating human Scot-t genomicDNA and protein together with SNPs positions. Horizontal arrows show twopairs of primers.

FIG. 2 shows a genomic DNA sequence of protamine-1 and primers for PCRamplification and sequence analysis. Translation starting point, intron,and canonical poly A addition signal are shown by +1, an outline box,and shadowed characters, respectively. The primer sequences areunderlined parts. The amino acid sequence of each protein is writtenwith capital letters under the nucleotide sequence. Numerals in rightmargin show position numbers of nucleotide and amino acid (thickletters) sequences (the nucleotide position number is a number from thetranslation starting point (+1) and note that it is different from SEQID NO:170). SNPs are shown by thick letters. Asterisk shows differenceof nucleotides possessed by 496 cases of human male patients of parentpopulation in which infertility and reproduction ability are proved,from GenBank registered sequence (EMBL/DDBJ/GenBank/Y00443, M29706,M60331, M60332).

FIG. 3 shows, just as FIG. 2 shows, a genomic DNA sequence ofprotamine-2 and primers for PCR amplification and sequence analysis.

BEST MODE FOR CARRYING OUT THE INVENTION

The mouse spermatogenesis genes group (MSGs) is an assembly of 89 genesin total wherein cDNAs synthesized from mRNAs transcribed from therespective genes have the base sequences represented by SEQ ID NOS:1 to89.

cDNAs can be identified by cloning methods of (a) to (c) as mentionedabove or a combination thereof. These methods are all known methods andcan be carried out by those skilled in the art without requiring undueexperiment. For example, basic procedure of operations of thesubtraction method in the method (c) is exemplified in general textbooks(Current Protocol in Molecular Biology 1:5.8.9-5.9.20, Green PublishingAssociate and Willey-Interscience, 1987-). Namely, (i) after total RNAsand mRNAs are extracted from testis of mature mouse havingspermatoblasts at all differential stages, e.g., 35 day-old mouse andpurified, cDNAs corresponding to mRNAs are synthesized to prepare a cDNAlibrary, and separately (ii) total RNAs and mRNAs are extracted fromtestis of immature mouse having undifferentiated spermatoblasts, forexample, 17 day-old mouse and purified and the mRNAs are, for example,labeled with biotin. Then, (iii) hybridization is carried out betweenthe above cDNA library and excess concentration of the biotin-labeledmRNAs, and hybrids formed from these both substances and remainingexcess biotin-labeled mRNAs are removed by agglomeration with avidin toprepare a subtracted cDNA library. Thereafter, (iv) from the abovesubtracted cDNA library, for example, by Northern blotting using totalRNAs of both 17 day-old and 35 day-old mice, cDNAs specifically reactiveto the total RNAs of 35 day-old mouse are selected and collected(cloned) and thereby MSG clones can be obtained. In this regard, whenthe above cDNA library and subtracted cDNA library are formed in a formof the resulting transformants by inserting and linking the cDNAs to avector and transferring it into a host Escherichia coli, subsequentamplification, preparation, and screening of the cDNAs are allfacilitated.

The MSGs of the invention can also be defined as genomic DNA fragmentswhich hybridize oligonucleotide probes synthesized based on the basesequence information of cDNA clones (SEQ ID NOS:1 to 89) identified bythe above methods. Hybridization is carried out under “stringentconditions” wherein salt concentration, organic solvent concentration,and temperature, and the like are within certain ranges.

Thus identified gene DNA (genomic DNA) of the MSGs can be isolated byscreening the mouse genomic DNA library which is cloned to a BAC(Bacterial Artificial Chromosome) vector, a cosmid vector, a phagevector, or the like, using the above probes. The isolated genomic DNAfragments can be also used as probes, for example, for diagnosingchromosome aberration by fluorescent in situ hybridization (FISH).

Moreover, determination of base sequence of the obtained MSGs clonecDNAs can be conducted by a known method and is usually conducted by acycle sequence method (Current Protocol in Molecular Biology, 1:7.4A.12-7.4A.13) using dideoxy method (Sanger method) as an elementarymethod. An advantage of the cycle sequence method is that apolynucleotide obtained from PCR amplification can be directlysequenced, which can be achieved using a commercially available ThermoSequenase fluorescent labelled primer cycle sequencing kit [manufacturedby Amasham (USA)] and a LC4000 autosequencer [manufactured by LI-COR].

In this regard, polynucleotides (DNA fragments and RNA fragments) can bealso purified by known methods from genomic DNAs or mRNAs of respectivegenes belonging to MSGs. Such purified polynucleotides are useful, forexample, as targets to be tested in the toxicity test and the like.

Furthermore, these polynucleotides and the above cDNAs can be used fordetection and cloning of homogeneous genes. Namely, for example,homology search using the base sequences of SEQ ID NOS:1 to 89 enablesdetection of homologous genes of animals and plants other than mousehomologous to the MSGs, e.g., homologous base sequences in human genomicDNA. For the homology search, it is possible to use gene databasesprovided by, for example, DDBJ (http://www.ddjb.nig.ac.jp/), NCBI(http://www/ncbi.nim.nih.gov/), and the like. The homologous gene can befound in the form of a full-length base sequence, EST (expressionsequence tags), STS (sequence tagged sites), GSS (genome surveysequence), SNP (single nucleotide polymorphism), or the like. Moreover,these homologous genes can be screened and cloned, for example, directlyfrom human chromosomal DNAs (using DNAs extracted and prepared fromchromosome of human sperm, leucocyte, etc.), or by hybridization usingprobes provided by the invention, RT-PCR with a PCR primer, or the like.

The DNA fragment group of the invention is an assembly of respectivegene DNAs belonging to the above MSGs group or continuous 10 to 99 basefragments (sense chains or antisense chains) of respective cDNAsbelonging to the above cDNA group. These DNA fragments are useful asprobes in the hybridization assay for detecting mutation of gene MSGs,for example. Moreover, they can be used as probes for the reproductiontoxicity test and preparation of a microarray for gene diagnosis ofinfertility.

These DNA fragments can be also prepared by synthesis in accordance withusual methods or optionally by cleavage of the above polynucleotides andcDNAs with appropriate restriction enzymes.

The primer set group of the invention is an assembly of syntheticoligonucleotides for PCR amplification of the above gene MSGs and cDNAs,and expression modulation and mutation of respective genes of MSGs canbe detected by PCR using these primer sets.

These primer sets can be designed on the basis of the base sequences ofSEQ ID NOS:1 to 89 and prepared via respective steps of synthesis andpurification. In this regard, since success of PCR largely depends onthe primer design and setting of various conditions, various ingenuityand trials are necessary to prepare an optimum primer. As points to keepin mind for the primer design, the following may be pointed out, forexample. The size of primer (number of bases) is from 15 to 40 bases,preferably 15 to 30 bases in consideration of satisfying a specificannealing with a template DNA. However, in the case that LA (longaccurate) PCR is carried out, the number is effectively at least 30bases. In order to prevent annealing of one set of the sense chain(5-terminal side) and the antisense chain (3-terminal side) or one pair(two primers) of primers each other, use of a complementary sequencebetween two primers is avoided. Also, in order to prevent formation ofhair-pin structure in the primers, it is tried to avoid use ofself-complementary sequence. Furthermore, in order to assure a stablebinding to the template DNA, GC content is made about 50% andlocalization of GC-rich or AT-rich may be avoided in the primers. Sinceannealing temperature depends on Tm (melting temperature), primershaving a Tm value which is from 55 to 65° C. and which is close to eachother is selected in order to obtain a PCR product having a highspecificity. Moreover, it is necessary to take note so that the finalconcentration of the primer used in the PCR becomes from about 0.1 toabout 1 μM. Furthermore, commercially available software for the primerdesign, for example, Oligo™ [manufactured by National Bioscience Inc.,(USA)], GENETYX [manufactured by Software Development K.K. (Japan)], orthe like may be employed. In this regard, the invention provides one ormore sets of two oligonucleotides for PCR amplification of each cDNA inrespective cDNA base sequences for SEQ ID NOS:1 to 89.

The microarray (DNA chip) of the invention is characterized in that theabove cDNAs or one or more of continuous 10 to 99 base fragments of thecDNAs are provided as probes in a substrate. In this regard, two or moreor five or more cDNAs are preferably provided. Such microarray may be amicroarray wherein the DNA fragments are directly synthesized on thesubstrate or a microarray wherein the DNA fragments are spotted on thesubstrate coated with a material with which DNA can combine.

The spermatogenic polypeptide group of the invention can be obtainedaccording to a method of isolation from mouse cells, a method ofpreparing peptides by chemical synthesis based on the amino acidsequences of SEQ ID NOS:90 to 167, or the like methods. In addition, thegroup can also be obtained in a large amount according to a geneticrecombination method using polynucleotides such as cDNAs. Namely,polypeptides encoded by MSGs can be obtained in a large amount byinserting cDNA or an ORF region thereof into an expression vector for invitro transcription or an expression vector suitable for prokaryoticcells such as Escherichia coli and Bacillus subtilis and eukaryoticcells such as yeasts, insect cells and mammalian cells, and by in vitrotranscription or from transformant cells transformed with the expressionvectors. The transformant cells can be produced by transferring therecombinant vectors into cells according to known methods such aselectroporation, calcium phosphate method, ribosome method, and DEAEdextran method. Moreover, for isolation and purification of thepolypeptides from culture products of the transformant cells, use can bemade of known methods, for example, treatment with a denaturing agentsuch as urea or a surfactant, ultrasonic treatment, enzymatic digestion,salting-out or solvent precipitation method, dialysis, centrifugation,ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusingelectrophoresis, ion-exchange chromatography, hydrophobicchromatography, affinity chromatography, reverse phase chromatography,and the like. In this regard, the polypeptides of the invention includefused proteins with other any proteins. For example, fused proteins withglutathion-S-tansferase (GST), green fluorescent protein (GFP), or thelike may be mentioned. Furthermore, proteins expressed in cells maysometimes undergo various modifications in the cells after translation.Accordingly, the modified proteins are also included in the invention.Such modifications after translation are elimination of N-terminalmethionine, N-terminal acetylation, sugar-chain addition, limiteddegradation with an intracellular protease, myristoylation,isoprenylation, phosphorylation, and the like.

The thus obtained polypeptides are useful as immunogens for antibodypreparation, target molecules for developing therapeutic agents forinfertility, or the like.

The antibody group of the invention is an assembly of polyclonalantibodies or monoclonal antibodies which recognize the above MSGspolypeptides. The antibody group is useful as materials for carrying outthe toxicity test and genetic diagnosis by investigating expression ofMSGs polypeptides or mutants thereof in spermatoblast. The antibodiesinclude all of the whole molecules capable of binding to epitopes ofMSGs polypeptides, and Fab, F(ab′)₂, Fv fragments, and the like. Forexample, in the case of polyclonal antibodies, such antibodies can beobtained from sera of animals after immunization of the animals usingthe above MSGs polypeptide or its partial peptide. Alternatively, theycan be prepared by collecting sera of animals after the above expressionvectors are transferred into muscle or skin of the animals by injectionor a gene gun. As the animals, mouse, rat, rabbit, goat, chicken, andthe like are used. Preparation of hybridomas by fusing B cells collectedfrom spleens of immunized animals with myelonas enables production ofmonoclonal antibodies.

The methods for toxicity test, mutagenicity test, and genetic diagnosisaccording to the invention can be carried out, for example, inaccordance with the following (A) to (I).

(A) Detection of ED (In Vivo)

For example, using an experimental animal such as mouse, guinea pig, ormonkey reared under administration of an ED-suspected substance,chromosomal DNA is extracted from the leukocytes, tissue cells, and thelike and purified (above Current Protocol in Molecular Biology1:2.2.1-2.2.3). Using the DNA, an experimental animal DNA homologous toMSGs is amplified by PCR with the above primer set to prepare an analyteDNA. The base sequence thereof is determined and homology search iscarried out between the analyte and normal gene DNA. As a result of thesearch, genetic diagnosis wherein mutation in the experimental animal,expression modulation induced by the mutation, and amino acidreplacement are analyzed can be carried out on the basis of theresulting difference in base sequence and amino acid sequence betweenboth DNAs. For the search, use can be made of, for example, acommercially available software for homology search (a program forhomology search provided by the above DDBJ or NCBI, e.g., FASTA, BLAST,PSI-BLAST, SSEARCH, etc.). In this regard, points to keep in mind forhomology search are described in, for example, a literature (CurrentProtocol in Molecular Biology 1:7.7.12-7.7.15). Furthermore, using RNAextracted from each organ (especially testis and sperm) of the aboveanimal, it can be judged whether the suspected substance is ED or not bydetecting change in expressed amount of mRNA by Northern blotting,RT-PCR method, microarray method, or the like with regard to MSGs.

(B) Detection of ED (In Vitro)

For example, using cell culture wherein an ED suspected substance isadded to and mixed with a medium and culture is effected, tetrahymena,echinus fertilized eggs, microorganisms, and the like, morphologicalanomaly (e.g., abnormal cell division, cellular degeneration, etc.)under the cultivation is detected on a microscope and also a gene DNA(analyte DNA) homologous to the MSGs is prepared from the above cultureproduct as in the above (A), followed by homology search. Thus, byinvestigating occurrence of mutation and change of expressed amount byNorthern blotting, RT-PCR method, or the like, it can be judged whetherthe suspected substance is ED or not. In this regard, as the culturecell, a transformant with an expression vector obtained by cloning eachpolynucleotide (cDNA) of the MSGs of the invention can be employed.

(C) Mutagenicity Test/Toxicity Test

Using new drugs under development and environmental pollution-suspectedchemicals as analytes, these tests can be carried out in a similarmanner to the above (A) and (B).

(D) Test of Influence on Reproduction (In Vivo)

Using new drugs under development and environmental pollution-suspectedchemicals as analytes, these tests can be carried out in a similarmanner to the above (A) and (B).

(E) Test of Influence on Reproduction (In Vivo)

An expression vector to which an MSG gene (polynucleotide such as cDNA)is inserted and linked is constructed and then transferred into a hostto prepare a transformant. The transformant is cultured in the presenceof a new drug under development or an environmental pollution-suspectedchemical. The present test can be carried out by judging whether theamount of its expression product or its function is normal or not. Forexample, Calmegin gene is expressed and the functional abnormality ofthe resulting Calmegin as shaperon is detected. Alternatively, by usingsuch an expression vector, it is possible to detect or search substanceswhich inhibit or accelerate its expressing ability and activity of anexpressed product.

(F) Preparation of Experimental Animal

For the purpose of carrying out analysis of in vivo functions of theMSGs and homologous genes thereof, animals wherein these genes areknocked out can be prepared. Moreover, for the purpose of analyses ofregulator gene loci of these genes and activity thereof and alsofunctions of gene products in individuals, transgenic animals can beprepared. Such experimental animals also enable development ofmedicaments and medical technologies for gene therapy and prevention ofmale infertility as well as preclinical test in development ofcontraceptives.

(G) Test with MSG-Transduced Transformant

As in the above (F), an expression vector of an analyte gene DNA isconstructed and its transformant is prepared. Then, abnormality offunctions of its expression products is detected and also mutation ofthe analyte DNA and amino acid replacement induced by the mutation areanalyzed. Contrastive analysis of both results thus obtained can createsmeanings of correlation between function of an expressed product and themutation.

(H) Amplified DNA by PCR

By PCR using the primers of the invention, DNAs of animals or culturedcells under various experimental conditions can be amplified using theseDNAs as templates. The basic procedure of PCR is described in, forexample, the above literature (Current Protocol in Molecular Biology1:15.0.1-15.3.8). Moreover, the experimental group DNAs amplified by PCRand their fragments or restriction enzyme fragments can be used foranalyses of SSCP (single strand conformation polymorphism), RFLP(restriction fragment length polymorphism), EST, STS, GSS, SNP, and thelike and for SAGE (serial analysis of gene expression). For example,they can be employed as analytes for polyacrylamide electrophoresis, asprobes for DNA microarrays or DNA chips, and as probes for hybridizationafter labeling.

The following will describe human male infertility gene mutations of theinvention.

The Scot-t mutant polynucleotide of the invention is a polynucleotidehaving any one or more mutations selected from the following:

-   -   “t” at 129th position is replaced by “c”;    -   “t” at 870th position is replaced by “g”;    -   “c” at 1071st position is replaced by “t”; and    -   “t” at 1667th position is replaced by “c”,        in the DNA sequence of SEQ ID NO:168 or a complementary sequence        thereof.

The Scot-t mutant polynucleotide can be isolated, for example, byscreening a cDNA library prepared from whole mRNAs of a male infertileperson using a Scot-t mutant oligonucleotide to be mentioned below as aprobe. Also, the polynucleotide can be isolated by RT-PCR with the totalmRNAs of a male infertile person as a template using the primer set ofthe invention to be mentioned below. Alternatively, it can be alsoobtained by transducing the above base replacement into wild-type Scot-tcDNA using a commercially available mutation kit or the like. The thusobtained cDNA can be amplified, for example, by conventional geneamplification methods such as PCR (polymerase Chain Reaction) method,NASBN (Nucleic acid sequence based amplification) method, TMA(Transcription-mediated amplification) method, and SDA (StandardDisplacement Amplification) method.

The Scot-t mutant polynucleotide of the invention can be employed in themethod for diagnosing male infertility according to the invention.Moreover, it is also used as a material for preparing the Scot-t mutantpolypeptide of the invention to be mentioned below in a gene engineeringmanner.

The Scot-t mutant oligonucleotide of the invention is an oligonucleotidewhich is part of the above Scot-t mutant polynucleotide and which iscomposed of a continuous DNA sequence of 10 to 99 containing eachmutation site, or a complementary sequence thereof.

The Scot-t mutant oligonucleotide can be prepared chemically by a knownmethod. Moreover, it can be also prepared by cleavage of the Scot-tmutant polynucleotide with an appropriate restriction enzyme.

The Scot-t mutant oligonucleotide can be also employed in the method fordiagnosing male infertility according to the invention. Alternatively,it is also used as a material for preparing the Scot-t mutantoligopeptide of the invention in a gene engineering manner.

The Scot-t mutant genomic polynucleotide of the invention is apolynucleotide (genomic DNA) derived from human chromosomal DNAs, whichhybridizes the Scot-t mutant polynucleotide or Scot-t mutantoligonucleotide or a complimentary sequence thereof under stringentconditions. The stringent conditions mentioned here are conditions whichenable a selective and detectable specific binding between thepolynucleotide or oligonucleotide and a genomic DNA derived from achromosome. The stringent conditions are defined by salt concentration,organic solvent (e.g., formamide), temperature, and the other knownconditions. Namely, stringency is increased by reducing the saltconcentration, increasing organic solvent concentration, or elevatinghybridization temperature. For example, a stringent salt concentrationis usually about 750 mM or less of NaCl and about 75 mM or less oftrisodium citrate, more preferably about 500 mM or less of NaCl andabout 50 mM or less of trisodium citrate, most preferably about 250 mMor less of NaCl and about 25 mM or less of trisodium citrate. Astringent organic solvent concentration is about 35% or more, mostpreferably about 50% or more of formamide. A stringent temperaturecondition is about 30° C. or higher, more preferably about 37° C. orhigher, most preferably about 42° C. or higher. The other conditionsinclude hybridization period of time, concentration of a washing agent(e.g., SDS), presence or absence of carrier DNA, and the like. Bycombining these conditions, various classes of stringency can be set. Asone preferable embodiment, hybridization is carried out at 30° C. underconditions of 750 mM of NaCl, 75 mM of trisodium citrate, and 1% of SDS.As a more preferable embodiment, hybridization is carried out at 37° C.under conditions of 500 mM of NaCl, 50 mM of trisodium citrate, 1% ofSDS, 35% of formamide, 100 μg/ml of denaturated salmon sperm DNA. As themost preferable embodiment, hybridization is carried out at 42° C. underconditions of 250 mM of NaCl, 25 mM of trisodium citrate, 1% of SDS, 50%of formamide, 200 μg/ml of denaturated salmon sperm DNA. In addition,conditions for washing after hybridization also affect stringency. Theconditions for washing are also defined by salt concentration andtemperature, and stringency at washing is increased by decrease of saltconcentration and elevation of temperature. For example, stringent saltconditions for washing are preferably about 30 mM or less of NaCl andabout 3 mM or less of trisodium citrate, most preferably about 15 mM orless of NaCl and about 1.5 mM or less of trisodium citrate. A stringenttemperature condition is about 25° C. or higher, more preferably about42° C. or higher, most preferably about 68° C. or higher. As onepreferable embodiment, washing is carried out at 25° C. under conditionsof 30 mM of NaCl, 3 mM of trisodium citrate, and 0.1% of SDS. As a morepreferable embodiment, washing is carried out at 42° C. under conditionsof 15 mM of NaCl, 1.5 mM of trisodium citrate, and 0.1% of SDS. As themost preferable embodiment, washing is carried out at 68° C. underconditions of 15 mM of NaCl, 1.5 mM of trisodium citrate, and 0.1% ofSDS.

The Scot-t mutant genomic polynucleotide can be isolated by screening agenome library, which is prepared from chromosomal DNAs of a maleinfertile person, by stringent hybridization as above using Scot-tmutant oligonucleotide as a probe and washing treatment.

The Scot-t mutant genomic polynucleotide may be a detection target inthe method for diagnosis according to the invention.

The Scot-t primer set of the invention is a primer set for PCRamplification of the Scot-t mutant polynucleotide, a double-strandedpolynucleotide composed of the Scot-t mutant genomic polynucleotide andits complementary sequence, or mRNA transcribed from the Scot-t mutantgenomic polynucleotide. In these primer sets, one oligonucleotide primeris composed of a continuous DNA sequence of 15 to 45 nt, preferably 15to 30 nt containing at least one nucleotide mutation site of SEQ IDNO:168 (Scot-t cDND) or a complementary sequence thereof. Another primermay be any continuous DNA sequence at 5′-side or 3′-side of eachmutation site of SEQ ID NO:168 or a complementary sequence thereof.

These primer sets can be prepared by a known process for DNA synthesisbased on SEQ ID NO:168 containing each mutation site. Moreover, at theterminal of the primer, a linker sequence or the like may be added.Furthermore, for designing the sequence, commercially availablesoftware, for example, Oligo™ [manufactured by National Bioscience Inc.,(USA)], GENETYX [manufactured by Software Development K.K. (Japan)], orthe like may be employed.

The Scot-t primer sets of the invention can be employed in the methodfor diagnosing male infertility according to the invention.

In the protamine-2 mutant polynucleotide of the invention, c at 248thposition in SEQ ID NO:173 (protamine-2 cDNA) is replaced by t.

The protamine-2 mutant oligonucleotide, mutant genomic polynucleotide,protamine-2 primer sets of the invention can be obtained and used in thesame manner as in the case of the above invention relating to Scot-t.

The Scot-t mutant polypeptide of the invention is a polypeptide which isan expression product of the Scot-t mutant polynucleotide or mutantgenomic polynucleotide of the above invention and which has one or moremutations selected from the following group:

-   -   Leu at 38th position is replaced by Pro;    -   Leu at 285th position is replaced by Arg; and    -   Thr at 352nd position is replaced by Met,        in the amino acid sequence of SEQ ID NO:169.

Namely, in these Scot-t mutant polypeptide, amino acids in the normal(wild-type) polypeptide (SEQ ID NO:169) are mutated as above by missensemutation in the Scot-t mutant polynucleotide of the above invention.

The protamine-2 mutant polypeptide is a short-chain polypeptide which isan expression product of the protamine-2 mutant polynucleotide of theabove invention and which is composed of an amino acid sequence of 1stto 49th positions in the amino acid sequence of SEQ ID NO:174. Namely,the polypeptide is a short-chain polypeptide which is formed by mutationof 50th glutamic acid codon into a stop codon by one base replacement (cby t) at 248th position of the protamine-2 mutant polynucleotide and asa result of no expression of the following protein-encoded region fromthe position.

These mutant polypeptides are obtained by a process for isolation from abiological sample of a male infertile person according to a knownmethod, a process for preparing peptides by chemical synthesis based onan amino acid sequence of SEQ ID NO:169 or 174 containing each mutantamino acid residue, or a process for production by a recombinant DNAtechnology using mutant polynucleotide (mutant cDNA) of the aboveinvention. These mutant polypeptides may be employed as test target inthe method for diagnosing male infertility according to the invention.

The mutant oligopeptides of the invention are oligopeptides which areparts of the respective mutant polypeptides of the above invention andwhich have an continuous amino acid sequence of 5 to 30 containing eachamino acid mutation site. These mutant oligopeptides can be prepared bya process of chemical synthesis based on a predetermined amino acidsequence or a process of digesting the above mutant polypeptides with anappropriate protease. These oligopeptides can be employed, for example,as antigens for antibody preparation according to the invention.

The anti-mutant Scot-t antibody, anti-mutant protamine-2 antibody, andanti-protamine-2 antibody of the invention are polyclonal antibodies ormonoclonal antibodies prepared using the oligopeptides of the aboveinvention as antigens and include all of whole molecules capable ofbinding to epitopes of the mutant polypeptides of the invention, andFab, F(ab′)₂, Fv fragments, and the like. Such antibodies can beprepared in a similar manner to the case of antibodies described for theMSGs invention. Moreover, these antibodies can specifically recognizethe above mutant polypeptides and thus employed in the methods fordiagnosis according to the invention.

The method for diagnosis according to the invention is a method fordiagnosing whether a subject person suffers from male infertility ornot. Namely, chromosomal DNAs are isolated from a biological sample ofthe subject person and, in the case that the mutant genomicpolynucleotide of the above invention is present in the DNAs, thesubject person is judged to be a high-risk person of male infertility.The subject person may be an infertile male person or a boy having aninfertile male person in relations on his mother's side, but is notlimited thereto. The mutant polynucleotide can be also detected by amethod of directly sequencing it but the following methods arepreferable.

First, it is detected whether chromosomal DNAs isolated from the subjectperson or mRNAs thereof hybridize the Scot-t mutant polynucleotideand/or protamine-2 mutant polynucleotide or respective mutantoligonucleotides under stringent conditions. In the case that thesubject person possesses a gene mutation associated with maleinfertility, the chromosomal DNAs or the mRNAs and mutantpolynucleotides or mutant oligonucleotides hybridize even understringent conditions. The hybridization can be detected by a knownmethod. For example, the detection can be carried out conveniently andat a high accuracy using the DNA probe or DNA chip of the invention. Asa hybridization method using a labeled DNA probe, specifically knownmethods, for example, Allele-specific Oligonucleotide Probe method,Oligonucleotide Ligation Assay method, Invader method, or the like canbe adopted. Moreover, the DNA chip may be a chip wherein the mutantpolynucleotides and/or mutant oligonucleotides are directly synthesizedon a substrate or a chip wherein the oligonucleotides are spotted on asubstrate coated with a material to which nucleotides may bind.Nucleotide mutation in a test sample DNA can be identified usingpresence of hybridization of the labeled test sample DNA and theoligonucleotides on the substrate as an indicator.

In the second preferable method, presence of a PCR product is detectedin the case that PCR is carried out with a primer set of the aboveinvention using chromosomal DNA or mRNA isolated from the subject personas a template. When the subject person possesses gene mutationassociated with male infertility, a PCR product of the polynucleotidedefined by the primer set is obtained. PCR or RT-PCR can be carried outby a known method. Nucleotide mutation may be detected by, other than amethod of direct sequencing of a PCR product, PCR-SSCP method, PCR-CFLPmethod, PCR-PHFA method, or the like. Moreover, a known method such asRolling Circle Amplification method or Primer Oligo Base Extensionmethod can be also employed.

In the application of another method for diagnosing male infertility ofthe invention, in the case that the Scot-t mutant polypeptide and/orprotamine-2 mutant polypeptide of the above invention is present in abiological sample isolated from a subject person, the subject person isjudged to be a high-risk person of male infertility. Although thepolypeptides can be detected by various known method, one preferablemethod is a method of detecting the Scot-t mutant polypeptide using ananti-mutant Scot-t antibody. Moreover, it is a method of combined use ofan anti-mutant protamine-2 antibody and an anti-protamine-2 antibody (anantibody prepared using an oligopeptide composed of an amino acidsequence of 50th to 91st positions in SEQ ID NO:7 or of 1st to 11thpositions in SEQ ID NO:8). Namely, in the case of short-chainprotamine-2 mutant polypeptide, the anti-mutant protamine-2 antibodyreacts but the anti-protamine-2 antibody prepared using long-chainprotamine-2 polypeptide does not react.

In the case of the method for diagnosis using the above antibodies, aconvenient and highly accurate detection is possible especially by usinga labeled antibody in this invention. For the labeling, an enzyme,radioactive isotope, or fluorescent dyestuff can be employed. The enzymeis not particularly limited as far as it satisfies requirements that ithas a large turnover number, it is stable even when combined with anantibody, it specifically colors a substrate, and the like. Use can bemade of enzymes usable for usual EIA, for example, peroxydase,β-galactosidase, alkaline phosphatase, glucose oxidase, acetylcholineesterase, glucose-6-phosphorylation dehydrogenase, malate dehydrogenase,and the like. Moreover, an enzyme-inhibiting substance, coenzyme, andthe like can be also employed. The combination of the enzymes with anantibody can be effected by a known method using a crosslinking agentsuch as maleimide compound. As the substrate, a known substance can beemployed in accordance with the kind of enzyme to be used. For example,3,3′,5,5′-tetramethylbenzidine can be used in the case that a peroxidaseis used as the enzyme, and p-nitrophenol can be used in the case thatalkaline phosphatase is used as the enzyme. As the radioisotope, thoseused in usual RIA, such as ¹²⁵I and ³H can be employed. As thefluorescent dyestuff, those used in usual fluorescence antibody methods,such as fluorescence isothiocyanate (FITC) and tetramethylrhodamineisothiocyanate (TRITC), can be used. In the case of using an enzyme,enzyme activity is determined by adding a substrate which is decomposedby the action of the enzyme to develop color and then opticallymeasuring the decomposed amount of the substrate, the activity isconverted into an amount of bound antibody, and an amount of theantibody is calculated based on the comparison with standard values. Inthe case of using a radioactive isotope, amount of radiation emitted bythe radioactive isotope is measured by a scintillation counter or thelike. Moreover, in the case of using a fluorescent dyestuff, an amountof fluorescence may be measured by a measuring equipment combined with afluorescent microscope. Furthermore, a sandwich method using a primaryantibody and a labeled secondary antibody (“ELISA method” in the case ofusing an enzyme as a label) can be also preferably employed.

EXAMPLES

The following will specifically describe embodiments and compositionsand advantages of the invention with reference to Examples and UseExamples but the invention of this application is not limited theseexamples.

Example 1 Preparation of Mouse Total RNA

Each testis was excised from the following two groups of mice (C57BL/6),19 mice of 17 days old and 5 mice of 35 days old and collected into onecapsule per group. Thereto was added 50 ml of 5.5M GTC solution (pH 7.5;5.5M guanidine thiocyanate, 25 mM sodium citrate 2H₂O, 0.5% (W/V) sodiumlauryl sarconate, and 0.2M 2-mercaptethanol), and then the mixture waspassed through a syringe with a 18 G needle to break the cells. Then,the mixture was subjected to low-speed centrifugation and thesupernatant was collected to remove precipitated cell fragments. Fiftymilliliters of the collected supernatant was transferred into two tubes,in which 14 ml of CsTFA (cesium trifluoroacetate) solution was placed,in an amount of 25 ml each, followed by ultra-centrifugation (25,000rpm, 15° C., 24 hours). RNA precipitated by the ultra-centrifugation wasdissolved in 600 μl of 4.4M GTC solution/tube and then precipitated withethanol. The precipitated RNA was dissolved in TE [10 mM Tris-HCl (pH7.5), 1 mM EDTA], and then recovered by re-precipitation with ethanol toobtain total RNA of each of both groups of 17 day-old and 35 day-oldmice.

Example 2 Preparation of Mouse mRNA[Poly(A)+]

Each of the total RNA in two tubes (two groups) in anethanol-precipitated state obtained in Example 1 was rinsed with 70%(V/V) ethanol, and then 500 μl of the above TE/tube was added todissolve the RNA, followed by heating at 65° C. for 5 minutes and rapidcooling on ice. Then, 500 μl of 1M NaCl/tube was added thereto and theresulting mixture was loaded on an oligo(dT)cellulose column [Type 3;manufactured by Collaborative Research] equilibrated with TE/NaCl (TE:1M NaCl=1:1) beforehand. Each column was washed with 8 ml of the aboveTE/NaCl and then each was eluted with 0.5 ml of TE to achievefractionation. This fractionation was repeated 5 times in total and aportion of each fraction was taken out and mixed with EtBr (ethidiumbromide). Thereafter, the second fraction from which fluorescentradiation was observed under UV irradiation was adopted as an mRNAfraction. On the second fraction, the above operations from heating at65° C. for 5 minutes to column fractionation was repeated again. Afterprecipitation of the resulting fraction with ethanol, the precipitatewas rinsed with 70% (V/V) ethanol and dissolved in 10 μl of TE. Aportion thereof was taken out and quantitative determination wasconducted on an absorptiometer.

Example 3 Preparation of cDNA Library of 35 Day-Old Mouse Testis

It was prepared according to the procedure described in the following(1) to (6).

(1) Synthesis of First Strand (Preparation of Single-Stranded, ss-cDNA)

Seven micrograms of mRNA of 35 day-old mouse obtained in Example 2 wasweighed out and distilled water was added thereto to make the totalamount 7.5 μl, followed by heating at 65° C. for 5 minutes and thencooling with ice. Thereafter, the following reagents were added andmixed: 2.5 μl of 10×1st strand buffer [500 mM Tris-HCl (pH 8.3), 750 mMKCl, 30 mM MgCl₂], 2.5 μl of 0.1M DTT (dithiothreitol), 1.5 μl of 1ststrand methyl nucleotide mixture (10 mM dATP, dGTP and dTTP; and 5 mM5-methyl-dCTP), 1 μl (1.6 μg) of linker primer [(GA)10ACGCGTCGACTCGAGCGGCCGCGGACCG(T) 18], 0.5M1 of RNase inhibitor, and 7.5μl of H₂O. The whole was left on standing at room temperature for 10minutes to effect annealing and then 2 μl of a reverse scriptase[manufactured by Seikagaku Corporation (Japan)] was added thereto,followed by reaction at 37° C. for 40 minutes. Then, 2 μl of SuperScript [manufactured by BRL (USA)] was added and mixed and then thewhole was allowed to react at 50° C. for 40 minutes to obtain asingle-stranded first strand (ss-cDNA).

(2) Synthesis of Second Strand (Preparation of Double-Stranded, ds-cDNA)

To the first strand solution obtained in the above (1) was added thefollowing reagents on ice and the whole was mixed: 20 μl of 10×2ndstrand buffer [188 mM Tris-HCl (pH 8.3), 906 mM KCl, 46 mM MgCl₂], 7.5μl of 0.1M DTT, 3 μl of 2nd strand nucleotide mixture (10 mM DATP, dGTPand dTTP; and 25 mM 5-methyl-dCTP), and 135 μl of H₂O. The whole wascooled with ice for 5 minutes and then 1.5 μl (2 units) of RNase H and 6μl (50 units) of Escherichia coli DNA polymerase I were added and mixed,followed by reaction at 16° C. for 180 minutes. After completion of thereaction, cDNA was extracted with 200 μl of phenol/chloroform(water-saturated phenol:chloroform=1:1 mixture) and chloroform,successively, and then was dissolved in 30 μl of 1/10 TE to obtain adouble-stranded second strand (ds-cDNA).

(3) Blunt End Formation (Preparation of Blunt End ds-cDNA)

To 30 μl of the ds-cDNA solution of the above (2) was added thefollowing reagents, and the whole was mixed: 10×T4 DNA polymerase buffer[500 mM Tris-HCl (pH 8.3), 100 mM MgCl₂, 500 mM NaCl, 100 mM DTT], 2.5mM dNTP mixture, 1 μl (10 units) of T4 DNA polymerase, and 54 μl of H₂O;the total amount was 100 μl. Then, after a reaction at 37° C. for 30minutes (keeping the period strictly), cDNA was extracted with 100 μl ofthe above phenol/chloroform and chloroform, successively, and then wasdissolved in 20 μl of 1/10 TE to obtain a blunt ended ds-cDNA.

(4) Linkage of Adaptor

To 4 μl of the blunt ended ds-cDNA solution of the above (3) was addedthe following reagents, and the whole was mixed and then cooled with icefor 5 minutes: 2 μl of 10× ligase buffer [500 mM Tris-HCl (pH 8.3), 70mM MgCl₂, 10 mM DTT], 2 μl of 10 mM ATP, 1 μl (0.35 μg) of an adaptor,and 9.5 μl of H₂O; the total amount was 18.5 μl. The above adaptor is a1:1 (W/W) mixture of BamH I(Bgl II)-Sma I[d(GATCCCCGGG)] [manufacturedby Takara Shuzo Co., Ltd.] and pSma I linker[d(pCCCGGG)] [manufacturedby Takara Shuzo Co., Ltd.] and was prepared by dissolving them in abuffer [10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10 mM MgCl₂] to have aconcentration of 0.35 μg/μl. Then, 1.5 μl (4 units) of T4 ligase wasadded and mixed. After a reaction at 8° C. overnight, the whole washeated at 70° C. for 30 minutes and then centrifuged at 15,000 rpm at 4°C. for 5 seconds, the supernatant being obtained as an adaptor-linkedds-cDNA.

(5) Preparation of Restriction Enzyme Fragments of ds-cDNA andFractionation by Spin Column

To the adaptor-linked ds-cDNA solution of the above (4) was added 27 μlof Not I buffer [278 mM NaCl, 8 mM MgCl₂, 1.8 mM DTT, 0.01% (W/V) BSA(bovine serum albumin), 0.018% (W/V) Triton X-100] and 3 μl of Not I (10units), respectively and the whole was mixed, followed by a reaction at37° C. for 150 minutes. After completion of the reaction, 5 μl of 10×STE[1M NaCl, 100 mM Tris-HCl (pH 8.3), 10 mM EDTA (pH 8.0)] and 10 μg oftRNA were added and mixed. The resulting mixture was placed in ChromaSpin columns [manufactured by Clontech (USA)] in an amount of 10μl/column and centrifuged at 4° C. at 1,800 rpm for 5 minutes and then afraction of restriction enzyme fragment at a bottom of the centrifugaltubes was collected. Then, the fragment was extracted with equivalentamount of phenol/chloroform and chloroform, successively, and then 10 μgof tRNA was replenished and the total amount is made 250 μl with addingwater. Furthermore, precipitation with ethanol was carried out and theprecipitate was rinsed with 70% (V/V) ethanol and then slightly dried toobtain a restriction enzyme (Not I/Bgl II) fragment of the adaptorlinked ds-αDNA.

(6) Insertion and Linkage of ds-Restriction Enzyme into a Vector

To the slightly dried product of the ds-DNA restriction enzyme (NotI/Bgl II) fragment of the above (5) was added the following reagents,and the whole was mixed and then cooled with ice for 5 minutes: 3 μl ofthe above 10× ligase buffer, 3 μl of 10 mM ATP, 1 μl (1 μg) of a plasmidvector pAP3neo (restriction enzyme Not I/Bgl II cleavage), and 22 μl ofH₂O. Then, 1 μl (4 units) of T4 DNA ligase was added and mixed. After areaction at 12° C. overnight, the whole was heated at 70° C. for 30minutes. After extraction with phenol/chloroform and chloroform,successively, the product was dissolved in 20 μl of TE to obtain asolution of a plasmid vector to which the ds-cDNA was inserted. Then,using the whole amount of the solution, ds-cDNA-inserted plasmid vectorwas transferred into a competent cell, Escherichia coli byelectroporation to effect transformation. The resulting transformant wasprovided as a cDNA library of 35 day-old mouse testis, for thepreparation of a subtracted library in Experiment 4 to be mentionedbelow.

Example 4 Preparation of Subtracted Library of Mouse

According to the procedures (1) to (5) described below, a subtractedlibrary was prepared by subtracting expression genes (mRNA:B) in 17day-old mouse testis from the cDNA library (A) of 35 day-old mousetestis obtained in Experimental Example 3 by hybridization. In order toremove also genes over expressed prior to the appearance of haploid bythe subtraction, the reactant ratio of A/B=1/40 was adopted in thehybridization.

(1) Conversion of cDNA Library (A) of 35 Day-Old Mouse Testis intoSingle-Stranded Ones

The transformant (Escherichia coli) of the cDNA library obtained in (6)of Experiment 3 was cultured at 37° C. for 1 hour in 4 ml of SOC [2%(W/V) Bacto-tryptone, 0.5% (W/V) yeast extract, 10 mM NaCl, 2.5 mM KCl,and 20 mM glucose] medium. Thereafter, it was transferred into 100 ml ofLB/Amp [1% (W/V) Bacto-tryptone, 0.5% (W/V) yeast extract, 0.5% (W/V)NaCl, 50 μg/μl ampicillin] medium and further cultured at 37° C. for 4.5hours to amplify the cDNA. Among 100 ml of the resulting culturesolution, 50 ml thereof was transferred into another vessel and 1 ml ofa helper phage (R408; 2×10¹² pfu (plaque-formation unit)/ml) was addedthereto, followed by culturing at 37° C. for 10 hours. The remaining 50ml of the culture solution was frozen and stored under addition andmixing of DMSO at a final concentration of 7% (V/V) after the culture at37° C. without further treatment. After completion of the culture, fungiwere removed by centrifugation and collected supernatant was filtratedthrough a filter having a pore size of 0.2 μm to remove Escherichia colidebris completely. Then, to 50 ml of the culture filtrate was added thefollowing reagents, and the whole was mixed: 5 ml of Dnase I solution[0.1M Tris-HCl (pH 7.5) and 0.1M MgCl₂] and 5 μl of Dnac I. The wholewas reacted at 37° C. for 30 minutes and then 20% (W/V) PEG(polyethylene glycol; solvent was 2.5M NaCl) was added to a quarter ofthe whole and mixed, followed by further reaction at room temperaturefor 20 minutes. Then, the phage was collected by ultracentrifugation at4° C. at 10,000 rpm for 10 minutes and, after supernatant was discardedand PEG was completely removed, the phage was suspended in 400 μl of TE.Thereto were added 25 μl of Proteinase K solution (solution composed of2 mg of Proteinase K, 800 μl of TE, and 200 μl of glycerol) and 4 μl of10% (W/V) SDS (sodium dodecyl sulfate), and the whole was mixed,followed by a reaction at 42° C. for 1 hour. After completion of thereaction, extraction was carried out with phenol, phenol/chloroform, andchloroform, successively, and precipitation with ethanol was carriedout. The precipitate was rinsed with 70% (V/V) ethanol and thendissolved in 100 μl of TE, a portion of which was quantitativelydetermined using a ultraviolet spectrometer.

(2) Biotinylation of 17 Day-Old Mouse Testis mRNA

Distilled water was added to a tube containing 40 μg of 17 day-old mousetestis mRNA obtained in Experimental Example 2, and the total amount wasmade 20 μl. Thereto was added 30 μl of biotin (photoprobe biotin 1 μg/μlaqueous solution)[manufactured by Vector Laboratories (USA)], followedby mixing. After thorough pipetting, the tube was placed on ice with thecap off and labeling was carried out by irradiating the tube with amercury lamp (BHRF100/110 v/60 W) from 10 cm above the tube for 20minutes. After completion of the labeling, 50 μl of 0.1M TE (pH 9.5) wasadded thereto and, after thorough pipetting, 100 μl of water-saturatedbutanol was added and excess of biotin was removed. Furthermore, 100 μlof chloroform was added to remove butanol, and then precipitation withethanol and rinsing with 70% (V/V) ethanol were successively carriedout, followed by dissolution into 20 μl of distilled water. The abovelabeling operation was repeated again and finally the product wasdissolved in 10 μl of distilled water to form a biotinylated mRNAaqueous solution.

(3) Hybridization

The following reagents were added to a PCR tube and mixed togethercarefully so as not to contain bubbles: 1.5 μl (0.5 μg) of ss-cDNAprepared in the above (1), 5 μl (20 μg) of the biotinylated mRNAobtained in (2), 12.5 μl of 2×HB [80% (W/V) formamido, 100 mM HEPES, 2mM EDTA, and 0.2% (W/V) SDS; prepared just before use], 2.5 μl of 2MNaCl, and 1 μl of Poly A (1 μg/μl aqueous solution) [manufactured byPharmacia (Sweden)], 2.5 μl of distilled water. It was reacted at 65° C.for 10 minutes and further at 42° C. for 43 hours to effecthybridization.

(4) Recovery of ss-cDNA by Reaction with Avidin

Into another tube was transferred 25 μl of the reaction solutionobtained in the above (3), and then 400 μl of SB [50 mM HEPES (pH 7.5),2 mM EDTA, and 500 mM NaCl], and 10 μg of streptoavidin [manufactured byGibco BRL (USA)] were added thereto and the whole was mixed. After areaction at room temperature for 5 minutes, ss-cDNA not hybridized wasextracted with 400 μl of phenol/chloroform (1:1 equivalent mixture). Theabove operations from the avidin-addition to extraction withphenol/chloroform was repeated again. Thereafter, further extractionwith chloroform was carried out and purified ss-cDNA was recovered bymillipore filter. Then, it was dissolved in 30 μl of 1/10 TE and then 15μl thereof was transferred into another tube (the remainder was storedat −20° C.) and concentrated (not completely dried) by vacuum drying for10 minutes.

Furthermore, the above operations from hybridization to vacuum dryingwere repeated twice. The composition at the second and thirdhybridization was as follows: ss-cDNA concentrated by the above vacuumdrying, 5 μl (10 μg) of the biotinylated mRNA, 12.5 μl of 2×HB, 2.5 μlof 2M NaCl, and 1 μl of Poly A, total amount of which was made 25 μl byadding distilled water. Moreover, of 30 μl of ss-cDNA obtained by secondhybridization, 15 μl was used for third hybridization and the remainderwas stored at −20° C.

(5) Conversion of ss-cDNA into Double-Stranded One

Fifteen microliters of PNK reaction mixture was added to and mixed with15 μl of the ss-cDNA solution obtained in the above (4), followed by areaction at 65° C. for 10 minutes. The PNK reaction mixture was preparedby reacting the following composition at 37° C. for 30 minutes: 1 μl ofprimer oligonucleotide for double strand formation, 3 μl of 10× ligationbuffer [500 mM Tris-HCl (pH 7.5), 70 mM MgCl₂, and 10 mM DTT], 3 μl of10 mM rATP, 2 μl of PNK (T4 polynucleotide kinase; 10 units/μl)[manufactured by Toyobo Co., Ltd. (Japan)], and 21 μl of distilled waterwere added and the total amount was made 30 μl. After completion of thereaction, the mixture was cooled to room temperature and then thefollowing reagents were added to 30 μl of the reaction solution and thewhole was mixed, followed by further reaction at 65° C. for 1 hour: 5 μlof 10× BcaBEST Buffer [manufactured by K.K. Takara (Japan)], 10 μl of 1mM dNTP, 0.5 μl of single strand DNA binding protein (2.1 μg/μl)[manufactured by USB (USA)], 2 μl of BcaBEST DNA polymeraze[manufactured by K.K. Takara (Japan)], and 3 μl of distilled water wereadded and the total amount was made 50 μl. After completion of thereaction, extraction with 100 μl of phenol/chloroform and chloroform wassuccessively carried out and purified ds-cDNA was recovered byfiltration through millipore filter. Then, it was dissolved in 20 μl ofTE and a portion thereof was transferred into Escherichia coli (XL-1Bleu) by electroporation to effect transformation. The thus obtainedtransformant was used for MSG cloning as a subtracted library, i.e.,MSG-candidate cDNA library.

Example 5 Cloning of MSG

Northern blotting was carried out between ss-cDNA prepared, in a similarmanner to the description in (1) of Experimental Example 4, from theculture solution of transformant containing the MSG-candidate cDNAlibrary of the subtracted library prepared in Experimental Example 4 andtotal RNA of both testis of 17 day-old and 35 day-old mice obtained inExperimental Example 1, and cDNA whose signal was detected only in 35day-old mouse was screened and collected. As a result, 79 MSG cloneswere obtained in total. Moreover, when clones obtained by cloning withmonoclonal antibody and polyclonal antibody were combined, 89 MSG cloneswere obtained in total.

Example 6 Determination of Base Sequence of MSG Clone DNA

After MSG clones/vectors were amplified by culturing respectivetransformant of MSG library obtained in Example 5, respective vectors(plasmids) were extracted and purified by alkali method. Then, based ondideoxy method (Sanger method), sequences of respective cDNAs of 89 MSGclones in total obtained in Example 1 were determined. The results areshown in SEQ ID NO:1 to 89.

Example 7 Investigation of ED Action Against MSG

As one model of ED discharged into the environment, change in expressionof MSG gene by administration of DES having estrogen action wasinvestigated using mouse.

To a 8 week-old C57BL/6 male mouse, DES was intraperitoneally injectedtwice every two days. After two days, both testes were taken out and onewas subjected to tissue observation while RNA was extracted from anotherone to compare a gene expression level.

Material: mouse: 8 week-old C57BL/6 male mice

Administration DES Concentration and Number of Mice

1: no treatment (two mice)

2: 0 μg DES in corn oil (two mice)

3: 1 μg DES in corn oil (two mice)

4: 10 μg DES in corn oil (two mice)

5: 50 μg DES in corn oil (three mice)

Experimental Schedule

On the first day, no treatment or each amount of DES dissolved in 20 μlof corn oil was intraperitoneally injected. On the third day, the sametreatment was conducted as in the first day. On the fourth day, testeswere taken out. One of them was fixed with Buan and embedded inparaffin, and then subjected to HE stain, followed by tissueobservation. Another one subjected to RNA extraction with Trizol and thegene expression level was compared by Northern hybridization.

Analyzed gene: OAZt (Ornithin decarboxylase antizyme-t): SEQ ID NO:50

Results

It was impossible to detect morphologically evident change. However, asa result of Northern hybridization on a spermatid-specificallyexpressing gene OAZt, individual difference was very large but, as awhole, the treated group showed a low level.

OAZ-t is a gene exhibiting a haploid spermatid-specific expression. Itwas suggested that initiation of the expression was suppressed by DES.In spit of such a low concentration and a short-term DES treatment thatno morphological anomaly was observed, difference was detected at a geneexpression level. This fact indicates that the method of the inventionis sufficiently applicable as a highly sensitive detecting system at anindividual level of influence of estrogen on male germ cells.

Example 8 Functional Analysis of Mouse Haspin

Haspin (SEQ ID NO:81) is a protein kinase which isspermatid-specifically expressed. It has various functional domain(nucleus-transferring signal, leucine zipper, transcription factor-likestructure) including a kinase domain and is involved in variousspermatid functions, so that it is considered to play important roles inspermatogenesis. Furthermore, when haspin gene is expressed in culturedcells, it is also known that the protein transfers into nuclei andgrowth of the cells was stopped at G1 stage (J. Biol. Chem. 274,17049-17057, 1999).

The following are revealed about the haspin.

Analysis of Hapsin-Knockout Mouse

Using thymidine kinase (TK) gene and neomycin (neo) resistance forselection, haspin gene of an ES cell was broken and a haspingene-defective (haspin KO) mouse was prepared by preparing mouse embryofrom the ES cell. The individual of the KO mouse normally developed andgrew but male mouse exhibited infertility and fertility was observed infemale mouse. Moreover, no apparent disorder was observed inspermatogenesis and morphologically not abnormal sperm was produced butit was confirmed that the mouse exhibited male infertility owing todysfunction.

Analysis of Expression Regulatory Region of Hapsin Gene

A transgenic (TG) mouse was prepared using a reporter gene wherein GFPgene was linked to 192 bases at upstream of haspin gene. As a result ofthe analysis of the TG mouse, GFP was expressed in a haploid spermatidspecific manner, so that it was confirmed that the region comprising the192 bases was a promoter which regulates the specific expression ofhaspin gene.

Analysis of Interactive Protein with Hapsin Molecule

From the experimental results of yeast 2 hybrid system, it was revealedthat there exist at least 8 kinds of intratesticular proteinsinteracting with haspin molecule. These proteins play important roles inspermatogenesis through interaction with human (Mol. Hum. Reprod. 7,211-218, 2001) or mouse haspin.

Based on these results, the following may be applicable. Namely, sinceit was confirmed that genetic deficit of haspin causes male infertility,

1) it is possible to cause male infertility by dysfunction of haspinitself or dysfunction of a protein interacting therewith;

2) it is possible to cause infertility by inhibiting interaction betweenhaspin and the other molecule; and

3) it is possible to cause infertility by inhibiting transfer of haspinto nucleus.

Namely, the inventors have found out a protein directing the specifictransfer to nucleus (importin alpha) among molecules interacting withhaspin. Dysfunction of the importin alpha can inhibit the transfer ofhaspin to nucleus and cause functional deficit of haspin.

4) it is possible to cause infertility by the action of an inhibitoragainst kinase activity of haspin.

Namely, since specific inhibitors against various kinases have hithertobeen developed, it is highly probable to develop an inhibitor against ahighly specific kinase such as haspin.

Moreover, the haspin promoter identified as above specifically expressesonly in haploid spermatid, but by activating the promoter in somaticcells, it is also possible to regulate the growth of abnormally growingcells. The activation of the promoter can be achieved by introduction ofa specific transcription factor effective thereon or a gene thereof.Alternatively, it can be achieved by a method of activating geneexpression of its transcription factor.

Example 9 Detection of ED in Mouse

A 10 day-old mouse (male) was reared for 200 days with continuousadministration of an ED-suspected analyte (Bisphenol A or the like)orally. Irrespective of observed anomaly on reproduction function,behavior, or appearance thereof, cellular RNA and chromosomal DNA wereextracted from the testis of the reared mouse using an RNA extractionkit (e.g., TOLIzol: GIBCO BRL) and a DNA extraction kit (e.g., DNAzol BDReagent: Oriental Yeast (Japan)). The RNA was investigated on change ofexpression level by Northern blotting and with regard to the DNA,amplification of spermatogenesis gene(s) was carried out using the PCRprimer obtained in Example 5. Thereby, when the amplification wasimpossible, it was judged that the gene(s) possess a large mutation.When the amplification is possible, the amplified DNA fragments weredirectly sequenced or, after fragmentation with an endonuclease whichrecognizes and cleaves a specific DNA base sequence, the fragments weresubjected to agarose gel or polyacrylamide gel electrophoresis and thenanalyses of SSCP, RFLP, EST, STS, GSS, and SNP were carried out todetect mutation. A DNA fragment in which mutation was detected wasfurther subjected to determination of its base sequence and then judgedon the following points by computer analysis: whether the mutation ispolymorphism or not; whether the mutation causes modulation during theprocess of regulation, duplication, and transcription of the gene(s);and whether the mutation induces a large disorder on the function oftranslation product(s) (protein(s)). Based on these change in RNA andDNA, ED was detected.

Example 10 Analysis of Human Scot-t Gene and Protamine Gene

1. Methods

1-1. Subject Person and Extraction of Genomic DNA

For the analysis of Scot-t gene, a total of 255 cases of maleinfertility patients were classified into a plurality of subordinategroups in accordance with spermatology. 152 cases (60%) werenonobstructive azoospermia, 72 cases (28%) were severe oligospermia(from 0.1 to 3×10⁶ cell/ml), 27 cases (11%) were low sperm motility, and4 cases (2%) were idiopathic infertility wherein number, morphology andmotility of sperm were normal. As a control group, the subjects were 261cases of reproducible male persons who were husbands of pregnant wivesvisited maternity hospitals.

For the analysis of protamine genes, a total of 258 cases of maleinfertility patients were classified into a plurality of subordinategroups in accordance with spermatology. 153 cases (59%) werenonobstructive azoospermia, 73 cases (28%) were severe oligospermia(number of sperm: less than 5×10⁶ cell/ml), 28 cases (11%) wereasthenozoospermia where sperm motility was low, and 4 cases (2%) wereidiopathic infertility wherein number, morphology and motility of spermwere normal. As a control group, the subjects were 270 cases of healthypersons as mentioned above.

Genomic DNA of each of the above infertile patients and healthy personswas extracted by a known method using a protease and phenol (Sambrookand Maniatis, in Molecular Cloning—A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York, 1989) and used.

1-2. Identification of Mutation in Human Scot-t Genomic DNA

Since human Scot-t contains a pseudo-gene having 19 bp deletion (18nucleotides of 745th to 762nd and one nucleotide of 778th intervening 15nucleotides) at the central part of a coding region (Tanaka H, et al.,Mol Human Reprod 2001; 8: 16-23), two kinds of PCR primers containingthe deleted region were prepared so that the pseudo-gene was notamplified. Namely, For the amplification of 5′ half, use were made of 25oligonucleotide (tgctctgtgacgcgcggcccgaggc: SEQ ID NO:176) at upstreamof presumed transcription initiation site and 26 oligonucleotide(cctccacgatctcttccacctccacc: SEQ ID NO:177) of from 770th to 745th, 24oligonucleotide (cggtggaggtggaagagatcgtgg: SEQ ID NO:183), and 25oligonucleotide (tccattcctcaccactgcacacctg: SEQ ID NO:178) at downstreamof presumed transcription unit (cf. FIG. 1).

Determination of the total sequence of the h-Scot-t DNA except for 35nucleotides located around the internal primer sequence (730-764) aroundthe central part was able with using 2 kinds of PCR amplificationfragments covering the left side and the right side of the n-Scot-t gene(FIG. 1).

1-3. Introduction of Various SNP Types of Genes into Recombinanth-Scot-t cDNA and Analysis of Succinyl CoA Tranferase Activity

On 72 hours after transfection of each cDNA using CaPO₄, HEK 293 cellwas lysed. Each cell lysis solution (5 mg protein) was subjected to achromatographic treatment using HPLC to separate into exogenous SCOT-tand endogenous SCOT-s, followed by analysis of enzyme activity.Standardization of succinyl CoA transferase activity was carried outusing a relative amount of h-SCOT-t protein evaluated on a densitometerfrom Western blotting signals using anti-SCOT-t antibody.

SCOT enzyme activity was carried out by a method described in aliterature (Marcondes, S, et al. (2001) Proc. Natl. Acad. Sci. USA. 98,7146-7151) with slight modification. Namely, content of the analytemixture were 50 mM Tris-HCl (pH 8.5), 0.2 mM succinyl CoA, 5 mM lithiumacetoacetate, 5 mM MgCl₂, 5 mM iodoacetamide, and an h-SCOT-t fraction.Spectroscopic analysis of SCOT activity was carried out by measuringformation of acetoacetyl-CoA absorption at 313 nm. Protein concentrationwas measured by Bradford method using bovine serum albumin as a standardsubstance.

1-4. Identification of Mutation of Protamine-1 and Protamine-2 GenomicDNA

Genomic DNA was isolated from blood by a known method using a proteaseand phenol. Two kinds of polymerase chain reaction (PCR) primer sets inboth of 5′ and 3′ lateral regions were constructed and genomic DNAs ofthese protamines were amplified. For protamine-1 (Domenjoud, L. et al.(1990) Genomics, 8, 127-133), 24 oligonucleotide (P1A;cccctggcatctataacaggccgc: SEQ ID NO:179) from −42 to −19 upstream fromthe transcription initiation site was used as the 5′-primer, and 24oligonucleotide (P1B; tcaagaacaaggagagaagagtgg: SEQ ID NO:180) from 492to 515 downstream from canonical PolyA addition signal AATAAA was usedas the 3′-primer. For protamine-2 (Domenjoud, L. et al. (1990) Genomics,8, 127-133), PCR amplification was carried out using 24 oligonucleotide(P2A; ctccagggcccactgcagcctcag: SEQ ID NO:181) from +49 to +72 as the5′-primer, and 24 oligonucleotide (P1B; gaattgctatggcctcacttggtg: SEQ IDNO:182) from 625 to 648 as the 3′-primer. By these primer setting, 557polynucleotide (SEQ ID NO:170) from −42 to 515 and 599 polynucleotidefrom 49 to 648 of protamine-1 and protamine-2 genes, respectively can beamplified in the PCR (FIG. 2). PCR conditions were as follows: forprotamine-1, 40 cycles of denaturation at 96° C. (45 seconds), annealingat 66° C. (45 seconds), and extension at 72° C. (1 minute) wereconducted, and for protamine-2, 40 cycles of denaturation at 98° C. (10seconds), annealing at 68° C. (45 seconds), and extension at 72° C. (45seconds) were conducted. The fragments amplified by PCR were purifiedusing SUPREC PCR spin column (Takara, Siga, Japan) and thermal cyclesequence analysis (ABI, CA, USA) was carried out. Determination of DNAsequences was carried out using the same PCR primers.

2. Results

2-1. Sequence Analysis of Human Scot-t DNA

By the PCR primer setting described in the above 1-2, pseudo-gene DNAwas not amplified and only true Scot-t genomic DNA was amplified, sothat male persons having a risk of infertility and control male personswhose reproducibility was proved were compared.

As a result, four sites of single nucleotide polymorphism: SNP wereobserved as shown in Table 2. In 516 cases of male persons in total of255 cases of infertile patients and 261 cases of volunteer persons whosereproducibility was proved, one site was present in the 3′-noncodingregion (t1667c), and the other sites were present in the coding regionand alterations at 38th, 285th and 352nd amino acids were induced (FIG.1). With regard to t129c SNP located at 38th amino acid (L38P) withinconsensus mitochondrial target sequence domain, in reproducible controlgroup and infertile patients, respectively, 94% (246 cases) and 96% (246cases) were major homozygous leucine type (t/t), 5.4% (14 cases) and2.7% (7 cases) were heterozygosity (t/c), and 0.4% (1 case) and 0.8% (2cases) were minor homozygosity, which was observed to cause an aminoacid alteration to proline (c/c). With regard to t870g SNP located at285th amino acid (L285R), in reproducible control group and infertilepatients, respectively, 80% (208 cases) and 80% (204 cases) were majorhomozygous leucine type (t/t), 19% (50 cases) and 15% (39 cases) wereheterozygosity (t/g), and 1.1% (3 cases) and 4.7% (12 cases) were SNPminor-type homozygosity (g/g), which was observed to cause an amino acidalteration to arginie (g/g). With regard to c1071t SNP located at 352thamino acid (T352M), in normal control group and infertile male persons,respectively, 96% (250 cases) and 93% (238 cases) were major homozygousleucine type (c/c), 3.1% (8 cases) and 4.3% (11 cases) wereheterozygosity (c/t), and 0.8% (2 cases) and 2.4% (6 cases) were minorhomozygosity (c/c), which was observed to cause an amino acid alterationfrom threonine (c/c) to methionine (t/t). With regard to the expressionrate of homozygous SNPs causing amino acid alterations at three regions,parent population of infertile patients exhibited significantly two tofour times higher value as compared with the value of the reproduciblecontrol group. Furthermore, in this case, very interesting SNP (t1667c)was found in the 3′-coding region. In normal control group and infertilemale parent population, respectively, 80% (206 cases) and 80% (200cases) were major t-type homozygosity (t/t), 19% (49 cases) and 15% (38cases) were heterozygosity (c/t), and 1.2% (3 cases) and 4.8% (12 cases)were c-type minor homozygosity (t/t). All the cases of the minorhomozygosity were double SNPs and in both of the normal reproduciblecontrol group (3 cases of male persons) and infertile patients (12 casesof male persons), respectively, genotype in t870g was minor g-typehomozygosity, but one exception was observed in the infertile cases (theabove were shown in Tables 3 and 4).

TABLE 3 Background of clinical survey for 255 cases of infertile malepersons and mutation in h-Scot-t gene (SNPs) Ratio(%) L38P L285R T352Mt1667c Azoospermia 152 (60) 2 6 3  6 Aevere oligospermia  72 (28) 0 5 2 5 Asthenozoospermia  27 (11) 0 1 1  1 Idiopathic infertility  4 (2) 0 00  0 255 (100) 2 12 6 12* Reproducible control 261 1 3 2  3** *Totalcase number: 250 cases (azoospermia 147 cases) **Total case number: 258cases

TABLE 4 Expression rate of 3 sites of SNPs in coding region and 1 sitein non-coding region in infertile and reproductive ability-proved parentpopulations Increase Type & ratio in position of Geno- ReproducibleInfertile infertile statistical SNPs type control case case significancet129c t/t 246(94) 246(96) (L38P) t/c  14(5.4)  7(2.7) c/c  1(0.4) 2(0.8) x2 p < 0.54 t870g t/t 208(80) 204(80) (L285) t/g  50(19)  39(15)g/g  3(1.1)  12(4.7) x4 p < 0.018 c1071t c/c 250(96) 238(93) (T352M) c/t 8(3.1)  11(4.3) t/t  2(0.8)  6(2.4) x3 p < 0.17 t1667c t/t 206(80)200(80) t/c  49(19)  38(15) c/c  3(1.2)  12(4.8) x4 p < 0.0182-2. Succinyl CoA Transferase Activity of Recombinant h-SCOT-t HavingSNPs

In order to investigate influence of SNPs on h-SCOT-t enzyme activity, arecombinant protein having 3 sites of SNPs inducing amino acidreplacement was expressed in HEK 293 cell and analysis of succinyl CoAtransferase activity was carried out in vitro. All the minor-type SNPslocated at t129c (L38P) and c1392t (T352M) exhibited a similar level ofenzyme activity to that of major-type SNPs. Contrarily, the minor-type(g/g) SNP located at T870G (L285R) diminished the enzyme activity byhalf in vitro as compared with the major-type (Table 5). Based on thisresult, it is shown that the succinyl CoA transferase activity ofh-SCOT-t is a prerequisite for male infertility and the SNPs diminishingthe enzyme activity contain some cause of male infertility.

TABLE 5 Sccinyl CoA transferase activity at inside of HEK 293 celltransformed using recombinant h-Scot-t cDNA having SNPs Major-type L38PL285R T352M Activity ratio 100+− 100+− 50+− 100+− Each assay wasconducted three times.2-3. Sequence Analysis of Human Protamine-1 DNA

A DNA fragment containing 557 bp amplified by PCR contained an introncomposed of 91 nucleotides of from 204th to 294th (SEQ ID NO:170, FIG.2). SNPs in 509 bp within each primer was identified by sequenceanalysis of a DNA fragment having 557 bp (FIG. 2). Since all DNA samplesamplified about the same amount of PCR products, it was confirmed thatthe SNPs were by no means contained in primer sequence region. Theexpression rate of SNP was evaluated on male infertile patients astargets, and was compared with the case of volunteers whose reproductiveability was proved. In a total of 528 cases of male subject persons(infertile patients 258 cases and target volunteers having reproductiveability 270 cases), SNPs were found at 5 sites and 4 sites of them werepresent in the 133th, 160th, 320th and 321st coding region and 1 sitewas present at 431st of 3′ non-translation region (FIG. 2 and Table 6).All the observed SNPs did not induce alteration of amino acid (silentmutation). All of the 3 sites of SNPs located at a133g, c160g, and g320aand SNP located at 14th and 46th amino acids (FIG. 2) were majorhomozygosity and heterozygosity and no minor homozygous SNP was observed(Table 6). With regard to the other c321a SNP located at 47th aminoacid, in infertile parent population and control parent populationhaving reproductive ability, respectively, 56.6% (146 cases) and 47.8%(129 cases) were homozygous major c/c type, 34.5% (89 cases) and 43.3%(117 cases) were heterozygosity (c/a), and 8.9% (23 cases) and 8.9% (24cases) were homozygous minor type (a/a) SNP. In addition to a431g SNP inthe 3′-non-coding region, all these SNP did not induce any amino acidalteration and also it was not shown that occurrence rate in infertilepatients was significantly higher than that in volunteers who wereproved to have reproductive ability (Table 6: the positions ofnucleotides in Table 6 corresponds to the positions of nucleotides inFIG. 2).

TABLE 6 Expression rate of SNPs in protamine-1 and protamine-2 genes ininfertile and reproductive ability-proved parent populations Number ofSNP (%) Position Reproductive Nucleo- Amino Geno- Infertilityability-proved tide Acid type group control Protamine-1 133 14(R) a/a250(96.9) 268(99.3) a/g  8(3.1)  2(0.7) 160 23(R) c/c 258(100) 269(99.6)c/a  0(0)  1(0.4) 320 46(R) g/g 257(99.6) 270(100) g/a  1(0.4)  0(0) 32147(R) c/c 146(56.6) 129(47.8) c/a  89(34.5) 117(43.3) a/a  23(8.9) 24(8.9) 431* a/a 257(99.6) 269(99) a/c  1(0.4)  1(1) Protamine-2 24850(Q) c/c 257(99.6) 270(100) (Ter)*** c/t  1(0.4)  0(0) 398** g/g148(57.4) 127(47.0) g/c  88(34.1) 118(43.7) g/a  0(0)  1(0.4) c/c 22(8.5)  24(8.9) 473** a/a 146(56.6) 127(47.0) a/c  90(34.9) 118(43.7)a/c  22(8.5)  25(9.3) Total 258 270 The meanings represented by addedasterisk symbol are as follows: *3′-non-coding region **Intron ***Ter:Stop codon (tag)2-4. Sequence Analysis of Human Protamine-2 DNA

SNPs in 551 bp within each primer were identified by sequence analysisof DNA fragment (SEQ ID NO:173) in 599 bp (FIG. 3). Since all PCRamplified about the same amount of DNA determined on agarose gelelectrophoresis, it was confirmed that the primer sequence region didnot contain SNPs. In this case, 3 sites of SNPs were observed in 599nucleotides in protamine-2 gene, and 1 site was present in exon and twosites in intron (FIG. 3). One site of heterozygous SNP in 248thnucleotide changed c into t, which changed glutamine into stop codon.This change was observed only one case of infertile patient among 153cases of azoospermia patients and also was not found inn 270 cases ofreproducible control parent population (Table 6). There is a possibilitythat this change is associated with a cause of azoospermia even inhemizygous state. Furthermore, 2 sites of SNPs of g398c and a473c wereobserved within intron. With regard to g398c, in infertile parentpopulation and reproducible control parent population, respectively,57.4% (148 cases) and 47.0% (127 cases) were major-type homozygosity,34.1% (88 cases) and 43.7% (118 cases) were heterozygosity (g/c), and8.5% (22 cases) and 8.9% (24 cases) were minor homozygous (c/c) type.Furthermore, the presence of different heterozygous SNP which was g/atype was observed in 1 case of the control group. With regard to anotherSNP of a473c, in infertile parent population and reproducible controlparent population, respectively, 56.6% (146 cases) and 47.0% (127 cases)were major-type homozygosity, 34.9% (90 cases) and 43.7% (118 cases)were heterozygosity, and 8.5% (22 cases) and 9.3% (25 cases) were c-typeminor homozygosity. There was no significant difference in expressionrate of these intron SNPs between in the case of infertile parentpopulation and in the case of reproductive ability-proved volunteers.

INDUSTRIAL APPLICABILITY

As precisely described in the above, the invention of this applicationprovides 89 clones of mouse spermatogenesis genes (MSG) and full-lengthcDNA base sequences of these MSGs. Moreover, the invention provides amethod for genetic diagnosis and methods for toxicity test andmutagenicity test using expression modulation and mutation of MSGs asmeasures. These test methods enable genetic diagnosis directly usingmale infertility gene DNA and analyses at a molecular level ofenvironmental toxicity as influence of minute amount of chemicalsubstances released into the environment on sexual differentiation andgerm cell differentiation and influence of substances havingreproduction toxicity and mutagenicity on the living body, especiallyinfluence on gene group exhibiting germ cell-specific expression.Furthermore, the invention contributes to detection and measurement ofED having a risk of inducing a low sperm count and reproductivedysfunction as well as global environmental assessment relating to ED.In addition, in the conventional test for “influence on reproduction”purposing detection of teratogenicity, the invention provides anadditional novel assay for “mutagenicity against MSGs” and henceremarkably contributes improvement of international standard forassuring safety of medicaments and chemicals.

Moreover, the invention provides Scot-t gene mutation and protamine-2gene mutation which cause hereditary male infertility. Furthermore, itprovides a method for diagnosing male infertility targeting these genemutations, mutant polypeptides derived from the gene mutations, and thelike.

1. A isolated polynucleotide which is the cDNA of human maleinfertility-associated gene Scot-t and consists of the nucleotidesequence of SEQ ID NO: 168, and has the following mutation: “t” at 870thposition is replaced by “g”.